Anti-pvrig antibodies and methods of use

ABSTRACT

The present invention is directed to anti-PVRIG antibodies and methods of using same.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/748,695, filed Jan. 21, 2020 which is a continuation of U.S. patentapplication Ser. No. 15/277,980, filed Sep. 27, 2016 which is adivisional of U.S. patent application Ser. No. 15/048,967, filed Feb.19, 2016, now U.S. Pat. No. 10,227,408, which claims priority under 35U.S.C. § 119 to U.S. Ser. No. 62/118,208, filed Feb. 19, 2015, and toU.S. Ser. No. 62/141,120, filed Mar. 31, 2015, and to U.S. Ser. No.62/235,823, filed Oct. 1, 2015, all of which are expressly incorporatedherein by reference in their entirety.

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 9, 2020, isnamed 114386-5002-US04_SL.txt and is 877,666 bytes in size.

BACKGROUND OF THE INVENTION

Naïve T cells must receive two independent signals fromantigen-presenting cells (APC) in order to become productivelyactivated. The first, Signal 1, is antigen-specific and occurs when Tcell antigen receptors encounter the appropriate antigen-MHC complex onthe APC. The fate of the immune response is determined by a second,antigen-independent signal (Signal 2) which is delivered through a Tcell costimulatory molecule that engages its APC-expressed ligand. Thissecond signal could be either stimulatory (positive costimulation) orinhibitory (negative costimulation or coinhibition). In the absence of acostimulatory signal, or in the presence of a coinhibitory signal,T-cell activation is impaired or aborted, which may lead to a state ofantigen-specific unresponsiveness (known as T-cell anergy), or mayresult in T-cell apoptotic death.

Costimulatory molecule pairs usually consist of ligands expressed onAPCs and their cognate receptors expressed on T cells. The prototypeligand/receptor pairs of costimulatory molecules are B7/CD28 andCD40/CD40L. The B7 family consists of structurally related, cell-surfaceprotein ligands, which may provide stimulatory or inhibitory input to animmune response. Members of the B7 family are structurally related, withthe extracellular domain containing at least one variable or constantimmunoglobulin domain.

Both positive and negative costimulatory signals play critical roles inthe regulation of cell-mediated immune responses, and molecules thatmediate these signals have proven to be effective targets forimmunomodulation. Based on this knowledge, several therapeuticapproaches that involve targeting of costimulatory molecules have beendeveloped, and were shown to be useful for prevention and treatment ofcancer by turning on, or preventing the turning off, of immune responsesin cancer patients and for prevention and treatment of autoimmunediseases and inflammatory diseases, as well as rejection of allogenictransplantation, each by turning off uncontrolled immune responses, orby induction of “off signal” by negative costimulation (or coinhibition)in subjects with these pathological conditions.

Manipulation of the signals delivered by B7 ligands has shown potentialin the treatment of autoimmunity, inflammatory diseases, and transplantrejection. Therapeutic strategies include blocking of costimulationusing monoclonal antibodies to the ligand or to the receptor of acostimulatory pair, or using soluble fusion proteins composed of thecostimulatory receptor that may bind and block its appropriate ligand.Another approach is induction of co-inhibition using soluble fusionprotein of an inhibitory ligand. These approaches rely, at leastpartially, on the eventual deletion of auto- or allo-reactive T cells(which are responsible for the pathogenic processes in autoimmunediseases or transplantation, respectively), presumably because in theabsence of costimulation (which induces cell survival genes) T cellsbecome highly susceptible to induction of apoptosis. Thus, novel agentsthat are capable of modulating costimulatory signals, withoutcompromising the immune system's ability to defend against pathogens,are highly advantageous for treatment and prevention of suchpathological conditions.

Costimulatory pathways play an important role in tumor development.Interestingly, tumors have been shown to evade immune destruction byimpeding T cell activation through inhibition of co-stimulatory factorsin the B7-CD28 and TNF families, as well as by attracting regulatory Tcells, which inhibit anti-tumor T cell responses (see Wang (2006),“Immune Suppression by Tumor Specific CD4⁺ Regulatory T cells inCancer”, Semin. Cancer. Biol. 16:73-79; Greenwald, et al. (2005), “TheB7 Family Revisited”, Ann. Rev. Immunol. 23:515-48; Watts (2005),“TNF/TNFR Family Members in Co-stimulation of T Cell Responses”, Ann.Rev. Immunol. 23:23-68; Sadum, et al., (2007) “Immune Signatures ofMurine and Human Cancers Reveal Unique Mechanisms of Tumor Escape andNew Targets for Cancer Immunotherapy”, Clin. Canc. Res. 13(13):4016-4025). Such tumor expressed co-stimulatory molecules have becomeattractive cancer biomarkers and may serve as tumor-associated antigens(TAAs). Furthermore, costimulatory pathways have been identified asimmunologic checkpoints that attenuate T cell dependent immuneresponses, both at the level of initiation and effector function withintumor metastases. As engineered cancer vaccines continue to improve, itis becoming clear that such immunologic checkpoints are a major barrierto the vaccines' ability to induce therapeutic anti-tumor responses. Inthat regard, costimulatory molecules can serve as adjuvants for active(vaccination) and passive (antibody-mediated) cancer immunotherapy,providing strategies to thwart immune tolerance and stimulate the immunesystem.

Over the past decade, agonists and/or antagonists to variouscostimulatory proteins have been developed for treating autoimmunediseases, graft rejection, allergy and cancer. For example, CTLA4-Ig(Abatacept, Orencia®) is approved for treatment of RA, mutated CTLA4-Ig(Belatacept, Nulojix®) for prevention of acute kidney transplantrejection and by the anti-CTLA4 antibody (Ipilimumab, Yervoy®), recentlyapproved for the treatment of melanoma. Other costimulation regulatorshave been approved, such as the anti-PD-1 antibodies of Merck(Keytruda®) and BMS (Opdivo®), have been approved for cancer treatmentsand are in testing for viral infections as well.

Accordingly, it is an object of the invention to provide PVRIGimmunomodulatory antibodies.

BRIEF SUMMARY OF THE INVENTION

Accordingly, it is an object of the invention to provide methods ofactivating cytotoxic T cells (CTLs) of a patient comprisingadministering an anti-PVRIG antibody to the patient, wherein a subset ofthe CTLs of the patient are activated.

It is a further object of the invention to provide methods of activatingNK cells of a patient comprising administering an anti-PVRIG antibody tothe patient, wherein a subset of the NK cells of the patient areactivated.

It is an additional object of the invention to provide methods ofactivating γδ T cells of a patient comprising administering ananti-PVRIG antibody to the patient, wherein a subset of the γδ T cellsof the patient are activated.

It is a further object of the invention to provide methods of activatingTh1 cells of a patient comprising administering an anti-PVRIG antibodyto the patient, wherein a subset of the Th1 cells of the patient areactivated.

It is an additional object of the invention to provide methods ofinhibiting the interaction of PVRIG and PVLR2 in a patient having acondition associated with this interaction comprising administering ananti-PVRIG antibody to the patient.

It is a further object of the invention to provide methods of treatingcancer in a patient, comprising administering an anti-PVRIG antibody tothe patient, wherein said cancer is treated.

It is an additional object of the invention to provide methods asoutlined above wherein the anti-PVRIG antibody comprises the vhCDR1,vhCDR2, vhCDR3, vlCDR1, vlCDR2 and vlCDR3 sequences from an antibodyselected from the group consisting of CPA.7.001, CPA.7.003, CPA.7.004,CPA.7.006, CPA.7.008, CPA.7.009, CPA.7.010, CPA.7.011, CPA.7.012,CPA.7.013, CPA.7.014, CPA.7.015, CPA.7.017, CPA.7.018, CPA.7.019,CPA.7.021, CPA.7.022, CPA.7.023, CPA.7.024, CPA.7.033, CPA.7.034,CPA.7.036, CPA.7.040, CPA.7.046, CPA.7.047, CPA.7.049, and CPA.7.050.

It is an additional object of the invention to provide methods asoutlined above wherein the anti-PVRIG antibody competes for binding withan antibody comprising the vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2 andvlCDR3 sequences from an antibody selected from the group consisting ofCPA.7.001, CPA.7.003, CPA.7.004, CPA.7.006, CPA.7.008, CPA.7.009,CPA.7.010, CPA.7.011, CPA.7.012, CPA.7.013, CPA.7.014, CPA.7.015,CPA.7.017, CPA.7.018, CPA.7.019, CPA.7.021, CPA.7.022, CPA.7.023,CPA.7.024, CPA.7.033, CPA.7.034, CPA.7.036, CPA.7.040, CPA.7.046,CPA.7.047, CPA.7.049, and CPA.7.050.

It is a further object of the invention to provide methods as outlinedabove wherein the anti-PVRIG antibody is selected from the groupconsisting of CPA.7.001, CPA.7.003, CPA.7.004, CPA.7.006, CPA.7.008,CPA.7.009, CPA.7.010, CPA.7.011, CPA.7.012, CPA.7.013, CPA.7.014,CPA.7.015, CPA.7.017, CPA.7.018, CPA.7.019, CPA.7.021, CPA.7.022,CPA.7.023, CPA.7.024, CPA.7.033, CPA.7.034, CPA.7.036, CPA.7.040,CPA.7.046, CPA.7.047, CPA.7.049, and CPA.7.050.

It is an additional object of the invention to provide methods asoutlined above wherein the anti-PVRIG antibody competes for binding withan antibody selected from the group consisting of CPA.7.001, CPA.7.003,CPA.7.004, CPA.7.006, CPA.7.008, CPA.7.009, CPA.7.010, CPA.7.011,CPA.7.012, CPA.7.013, CPA.7.014, CPA.7.015, CPA.7.017, CPA.7.018,CPA.7.019, CPA.7.021, CPA.7.022, CPA.7.023, CPA.7.024, CPA.7.033,CPA.7.034, CPA.7.036, CPA.7.040, CPA.7.046, CPA.7.047, CPA.7.049, andCPA.7.050.

It is a further object of the invention to provide methods as outlinedabove wherein the anti-PVRIG antibody comprises the vhCDR1, vhCDR2,vhCDR3, vlCDR1, vlCDR2 and vlCDR3 sequences from an antibody selectedfrom the group consisting of CHA.7.502, CHA.7.503, CHA.7.506, CHA.7.508,CHA.7.510, CHA.7.512, CHA.7.514, CHA.7.516, CHA.7.518, CHA.7.520.1,CHA.7.520.2, CHA.7.522, CHA.7.524, CHA.7.526, CHA.7.527, CHA.7.528,CHA.7.530, CHA.7.534, CHA.7.535, CHA.7.537, CHA.7.538.1, CHA.7.538.2,CHA.7.543, CHA.7.544, CHA.7.545, CHA.7.546, CHA.7.547, CHA.7.548,CHA.7.549, CHA.7.550, CPA.7.001, CPA.7.003, CPA.7.004, CPA.7.006,CPA.7.008, CPA.7.009, CPA.7.010, CPA.7.011, CPA.7.012, CPA.7.013,CPA.7.014, CPA.7.015, CPA.7.017, CPA.7.018, CPA.7.019, CPA.7.021,CPA.7.022, CPA.7.023, CPA.7.024, CPA.7.033, CPA.7.034, CPA.7.036,CPA.7.040, CPA.7.046, CPA.7.047, CPA.7.049, and CPA.7.050.

It is an additional object of the invention to provide methods asoutlined above wherein said the-PVRIG antibody competes for binding withan antibody selected from the group consisting of an anti-PVRIG antibodycomprising the vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2 and vlCDR3sequences from an antibody selected from the group consisting ofCHA.7.502, CHA.7.503, CHA.7.506, CHA.7.508, CHA.7.510, CHA.7.512,CHA.7.514, CHA.7.516, CHA.7.518, CHA.7.520.1, CHA.7.520.2, CHA.7.522,CHA.7.524, CHA.7.526, CHA.7.527, CHA.7.528, CHA.7.530, CHA.7.534,CHA.7.535, CHA.7.537, CHA.7.538.1, CHA.7.538.2, CHA.7.543, CHA.7.544,CHA.7.545, CHA.7.546, CHA.7.547, CHA.7.548, CHA.7.549, CHA.7.550,CPA.7.001, CPA.7.003, CPA.7.004, CPA.7.006, CPA.7.008, CPA.7.009,CPA.7.010, CPA.7.011, CPA.7.012, CPA.7.013, CPA.7.014, CPA.7.015,CPA.7.017, CPA.7.018, CPA.7.019, CPA.7.021, CPA.7.022, CPA.7.023,CPA.7.024, CPA.7.033, CPA.7.034, CPA.7.036, CPA.7.040, CPA.7.046,CPA.7.047, CPA.7.049, and CPA.7.050.

It is a further object of the invention to provide methods of diagnosingcancer comprising a) contacting a tissue from a patient with ananti-PVRIG antibody; and b) determining the presence of over-expressionof PVRIG in the tissue as an indication of the presence of cancer. Theanti-PVRIG antibody can be as described herein and as outlined above.

It is an additional object of the invention to provide antigen bindingdomains, including antibodies, which are anti-PVRIG antibodies,comprising the vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2 and vlCDR3sequences from an antibody selected from the group consisting ofCPA.7.001, CPA.7.003, CPA.7.004, CPA.7.006, CPA.7.008, CPA.7.009,CPA.7.010, CPA.7.011, CPA.7.012, CPA.7.013, CPA.7.014, CPA.7.015,CPA.7.017, CPA.7.018, CPA.7.019, CPA.7.021, CPA.7.022, CPA.7.023,CPA.7.024, CPA.7.033, CPA.7.034, CPA.7.036, CPA.7.040, CPA.7.046,CPA.7.047, CPA.7.049, and CPA.7.050.

It is a further object of the invention to provide anti-PVRIG antigenbinding domains (including antibodies) compositions that are anti-PVRIGantibodies, selected from the group consisting of CPA.7.001, CPA.7.003,CPA.7.004, CPA.7.006, CPA.7.008, CPA.7.009, CPA.7.010, CPA.7.011,CPA.7.012, CPA.7.013, CPA.7.014, CPA.7.015, CPA.7.017, CPA.7.018,CPA.7.019, CPA.7.021, CPA.7.022, CPA.7.023, CPA.7.024, CPA.7.033,CPA.7.034, CPA.7.036, CPA.7.040, CPA.7.046, CPA.7.047, CPA.7.049, andCPA.7.050.

It is a further object of the invention to provide anti-PVRIG antigenbinding domains (including antibodies) compositions that are anti-PVRIGantibodies, selected from the group consisting of h518-1, h518-2,h518-3, h518-4, h518-5, h524-1, h524-2, h524-3, h524-4, h530-1, h530-2,h530-3, h530-4, h530-5, h538.1-1, h538.1-2, h538.1-3, h538.1-4,h538.2-1, h538.2-2, and h538.2-3 (as depicted in FIG. 90).

It is an additional object of the invention to provide antigen bindingdomains, including antibodies, which are anti-PVRIG antibodies,comprising the vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2 and vlCDR3sequences from an antibody selected from the group consisting ofCHA.7.502, CHA.7.503, CHA.7.506, CHA.7.508, CHA.7.510, CHA.7.512,CHA.7.514, CHA.7.516, CHA.7.518, CHA.7.520.1, CHA.7.520.2, CHA.7.522,CHA.7.524, CHA.7.526, CHA.7.527, CHA.7.528, CHA.7.530, CHA.7.534,CHA.7.535, CHA.7.537, CHA.7.538.1, CHA.7.538.2, CHA.7.543, CHA.7.544,CHA.7.545, CHA.7.546, CHA.7.547, CHA.7.548, CHA.7.549, CHA.7.550,CPA.7.001, CPA.7.003, CPA.7.004, CPA.7.006, CPA.7.008, CPA.7.009,CPA.7.010, CPA.7.011, CPA.7.012, CPA.7.013, CPA.7.014, CPA.7.015,CPA.7.017, CPA.7.018, CPA.7.019, CPA.7.021, CPA.7.022, CPA.7.023,CPA.7.024, CPA.7.033, CPA.7.034, CPA.7.036, CPA.7.040, CPA.7.046,CPA.7.047, CPA.7.049, and CPA.7.050.

It is a further object of the invention to provide nucleic acidcompositions comprising: a) a first nucleic acid encoding the a heavychain variable domain comprising the vhCDR1, vhCDR2 and vhCDR3 from anantibody; and b) a second nucleic acid encoding a light chain variabledomain comprising vlCDR1, vlCDR2 and and vlCDR3 from an antibody. Theantibody is selected from the group consisting of CPA.7.001, CPA.7.003,CPA.7.004, CPA.7.006, CPA.7.008, CPA.7.009, CPA.7.010, CPA.7.011,CPA.7.012, CPA.7.013, CPA.7.014, CPA.7.015, CPA.7.017, CPA.7.018,CPA.7.019, CPA.7.021, CPA.7.022, CPA.7.023, CPA.7.024, CPA.7.033,CPA.7.034, CPA.7.036, CPA.7.040, CPA.7.046, CPA.7.047, CPA.7.049,CPA.7.050, CHA.7.502, CHA.7.503, CHA.7.506, CHA.7.508, CHA.7.510,CHA.7.512, CHA.7.514, CHA.7.516, CHA.7.518, CHA.7.520.1, CHA.7.520.2,CHA.7.522, CHA.7.524, CHA.7.526, CHA.7.527, CHA.7.528, CHA.7.530,CHA.7.534, CHA.7.535, CHA.7.537, CHA.7.538.1, CHA.7.538.2, CHA.7.543,CHA.7.544, CHA.7.545, CHA.7.546, CHA.7.547, CHA.7.548, CHA.7.549,CHA.7.550, CPA.7.001, CPA.7.003, CPA.7.004, CPA.7.006, CPA.7.008,CPA.7.009, CPA.7.010, CPA.7.011, CPA.7.012, CPA.7.013, CPA.7.014,CPA.7.015, CPA.7.017, CPA.7.018, CPA.7.019, CPA.7.021, CPA.7.022,CPA.7.023, CPA.7.024, CPA.7.033, CPA.7.034, CPA.7.036, CPA.7.040,CPA.7.046, CPA.7.047, CPA.7.049, and CPA.7.050.

It is an additional object of the invention to provide expression vectorcompositions comprising the first and second nucleic acids as outlinedherein and above.

It is a further object of the invention to provide host cells comprisingthe expression vector compositions, either as single expression vectorsor two expression vectors.

It is an additional object of the invention to provide methods of makingan anti-PVRIG antibody comprising a) culturing a host cell of theinvention with expression vector(s) under conditions wherein theantibody is produced; and b) recovering the antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Schematic presentation of the mechanisms of action of theinvention.

FIG. 2 presents mRNA Expression of PVRIG in various normal humantissues.

FIG. 3 presents mRNA expression of PVRIG in various immune populationderived from peripheral blood and bone marrow (based on GSE49910).

FIG. 4 presents mRNA expression of PVRIG in various CD3+ lymphocytepopulation (based on GSE47855).

FIGS. 5A, 5B and 5C presents mRNA expression of PVRIG in specific cellpopulations. FIG. 5A resents mRNA expression of PVRIG in specific cellpopulations obtained by laser capture microscopy (based on GSE39397).FIG. 5B presents mRNA expression of PVRIG in CD4 T-cells from normal andcancer patient as well as expression form CD4 T-cell expression fromdraining lymph nodes and TILs form breast cancer patients (based onGSE36765). FIG. 5C presents mRNA expression of PVRIG from CD8 and CD4T-cells derived from follicular lymphoma tumor and tonsil (based onGSE27928).

FIG. 6 presents PVRIG expression in normal tissues based on GTEx.Expression levels are shown in log 2(RPKM) values (fragments identifiedper million reads per kilobase). Values above 1 are considered highexpression. Tissues are ranked from top to bottom by the medianexpression. Each dot on the plot represent a single sample.

FIG. 7 presents PVRIG expression in cancerous tissues based on TCGA.Expression levels are shown in log 2(RPKM) values (fragments identifiedper million reads per kilobase). Values above 1 are considered highexpression. Tissues are ranked from top to bottom by the medianexpression. Each dot on the plot represent a single sample

FIG. 8 shows a heatmap representation of the enrichment analysis resultsin three categories: protein interactions, pathways and diseaseassociations. Results are ranked from top to bottom by average p-valueper row. Only the top 10 results from each category are shown. Graysquares indicate p-values<0.05. Each column in the heatmap correspondsto a normal or cancer tissue from which a list of highly correlatedgenes was derived (r>0.55 using at least 50 samples). As shown in theheatmap, PVRIG correlates with a T cell gene expression signature whichis strongly associated with the immune response and immune diseases.

FIG. 9 presents PVRIG expression in normal skin vs. melanoma (GTEx andTCGA analysis). Such over-expression was observed in additional solidtumors and results from infiltrating lymphocytes and NK cells in thetumor microenvironment. In normal condictions, no infiltrating immunecells are present and therefore PVRIG expression levels are very low.

FIG. 10 presents the correlations of PVRIG and PD1 in melanoma from TCGAsamples, with several T cell makers in lung adenocarcinoma, colonadenocarcinoma and melanoma. The marker CD3 is a general markers for Tcells and is also expressed on NKT cells. CD4 and CD8 markers are usedto characterized subpopulation of T cells.

FIG. 11 shows expression of PVRIG on human PBLs. Human PBLs derived fromtwo donors were evaluated for PVRIG expression. Both donor 61 and donor40 showed significant staining with anti-PVRIG specific Ab.

FIG. 12 shows PVRIG-Ig exhibits strong binding to all four humanmelanoma cell lines MEL-23, Mel-624 and Mel-624.38 and mel-888 tested.Binding is not affected by co-culture with engineered melanoma specificT cells. Grey line corresponds to isotype control, solid black linecorresponds to PVRIG-ECD-Ig.

FIG. 13 Correlation of PVRIG with T cells and subpopulations of T cells.CD3G is component of the T cell receptor complex, CD4 is a maker for Thelper cells and CD8A is component of CD8 protein used to identifycytotoxic T cells. PVRIG highly correlated with T cells in many types oftumors including lung adenocarcinoma, colon adenocarcinoma and melanomawhich are shown here.

FIG. 14 presents representative images from the Confirmation/Specificityscreen. All hits from the Primary screen, and EGFR-expressing vector(negative control), were re-arrayed/expressed in duplicate and probedwith PVRIG at 20 μg/ml. A specific hit with strong intensity is shown ingreen (PVRL2). Non-specific hits are shown in black. Another weak hit(MAG) was later shown to bind also other ligands, thus suggesting thatit is not specific.

FIGS. 15A-15E presents effect of various PVRIG-ECD-Ig M:M proteins onmouse CD4 T cell activation. Plates were coated with anti-CD3 mAb (2g/mL) in the presence of 10 g/ml PVRIG-ECD Ig (batch #198) or controlmIgG2a as described in materials and methods. Wells were plated with1×10⁵ CD4+CD25− mouse T cells per well in the presence of 2 μg/ml ofsoluble anti-CD28. (A) The expression of CD69 was analyzed by flowcytometry at 48 h post-stimulation, representative histograms are shown.Each bar is the mean of duplicate cultures, the error bars indicatingthe standard deviation. (B-C) Culture supernatants were collected at 48h post-stimulation and mouse IL-2 and IFNγ levels were analyzed byELISA. Results are shown as Mean±Standard errors of duplicate samples.(D) Dose response effect of immobilized PVRIG-ECD Ig (FIG. 92BB onsurface CD69 (D) and IFNγ secretion (E) is presented. Each bar is themean of triplicate cultures, the error bars indicating the standarderrors.

FIG. 16 presents FACS analysis on PVRIG transduced PBLs using a specificantibody. The percent of cells staining positive (relative to emptyvector transduced) for the protein is provided.

FIG. 17 presents FACS analysis on PVRIG (either co-expressed with F4 TCRor in a bi-cystronic vector with F4 TCR and NGFR transduced PBLs using aspecific antibody. The percent of cells staining positive (relative toempty vector transduced) for the protein is provided.

FIGS. 18A-18B presents FACS analysis performed on TCR transducedstimulated PBLs for experiment 1 (FIG. 18A) and in experiment 2 (FIG.18B) using a specific monoclonal antibody that recognizes theextra-cellular domain of the beta-chain from the transduced specificTCR. The percentage of cells staining positive is provided.

FIG. 19 shows expression of PVRIG on F4 expressing PBLs causes areduction of IFNγ secretion upon co-culture with SK-MEL23, MEL-624 andMEL-624.38 in comparison to expression of an empty vector.

FIGS. 20A-20B shows expression of PVRIG and F4 in PBLs byco-transduction (FIG. 20A) does not affect IFNγ secretion in co-culturewith melanoma cell lines. Expression of PVRIG and F4 in PBLs using abi-cystronic vector (FIG. 20B) causes a reduction of IFNγ secretion uponco-culture with SK-MEL23, MEL-624 and MEL-624.38 in comparison toexpression of an empty vector.

FIG. 21 shows expression of PVRIG and F4 in PBLs using a bi-cystronicvector causes a reduction in T cell mediated cytotoxicity uponco-culture with melanoma cell lines.

FIG. 22 shows PVRIG expression in 3 subgroups of low, no change and highlevels of exhausted T cells. Exhausted T cells were selected based onhigh level expression of 4 markers: CD8A, PD-1, TIM-3 and TIGIT. Lowexpressing samples are not shown since none had any detectable levels ofPVRIG.

FIGS. 23A-23B: Western blot analysis of ectopically expressed humanPVRIG protein. Whole cell extracts of HEK293 cell pools, previouslytransfected with expression construct encoding human PVRIG-flag (lane 2)or with empty vector (lane 1) were analyzed by WB using an anti-flagantibody (23A) or anti-PVRIG antibodies (23B).

FIG. 24: Cell surface expression of HEK293 cells ectopically expressedhuman PVRIG-flag protein by FACS analysis. Anti-PVRIG pAb (Abnova) wasused to analyze HEK293 cells stably expressing the human PVRIG-flagprotein. Cells expressing the empty vector were used as negativecontrol. Detection was carried out by Goat Anti-mouse PE-conjugatedsecondary Ab and analyzed by FACS.

FIG. 25 depicts the full length sequence of human PVRIG (showing twodifferent methionine starting points) and the PVRIG Fc fusion proteinused in the Examples. The signal peptide is underlined, the ECD isdouble underlined, and the Fc domain is the dotted underlining. PVRIG,also called Poliovirus Receptor Related Immunoglobulin Domain ContainingProtein, Q6DKI7 or C7orf15, relates to amino acid and nucleic acidsequences shown in RefSeq accession identifier NP_076975, shown in FIG.25

FIG. 26 depicts the sequence of the human Poliovirus receptor-related 2protein (PVLR2, also known as nectin-2, CD112 or herpesvirus entrymediator B, (HVEB)), the binding partner of PVRIG as shown in Example 5.PVLR2 is a human plasma membrane glycoprotein.

FIG. 27 PVRIG antibody specificity towards HEK cells engineered tooverexpress PVRIG. Data shows absolute geometric MFI (gMFI) measurementsas a function of increasing antibody concentration. The broken blackline with squares shows staining of HEK hPVRIG cells with arepresentative anti-human PVRIG antibody (CPA.7.021), and the solidblack line with circles shows staining of HEK parental cells with thesame antibody.

FIG. 28 PVRIG RNA was assessed in various cancer cell lines by qPCR.Data shown is relative expression of PVRIG RNA in cell lines as foldchange over levels in expi cells as assessed by the 2^((−ΔΔCt)) method.

FIG. 29 PVRIG RNA was assessed in sorted PBMC subsets by qPCR. Datashown is relative expression of PVRIG RNA in each subset as fold changeover levels in HEK GFP cells as assessed by the 2^((−ΔΔCt)) method.D47-D49 denote three individual donors. CD4 denotes CD4 T cells, CD8denotes CD8 T cells, CD14 denotes monocytes, and CD56 denotes NK cells.

FIGS. 30A-30B. FIG. 30A: PVRIG RNA was assessed in sorted CD4 T cells(CD4) and NK cells (NK) under naïve and activated conditions by qPCR.CD4 T cells were stimulated with human T cell stimulator dynabeads and50 U/ml IL-2 for 3 days. NK cells were stimulated in 50 U/ml IL-2 for 3days. Data shown is relative expression of PVRIG RNA in each subset asfold change over levels in expi cells as assessed by the 2^((−ΔΔCt))method. Jurkat is included as a positive control. D47-D49 denote threeindividual donors. FIG. 30B PVRIG RNA was assessed in sorted CD8 T cellsunder naïve and activated conditions by qPCR. CD8 T cells werestimulated with human T cell stimulator dynabeads and 100 U/ml IL-2 for3 days. Data shown is relative expression of PVRIG RNA in each subset asfold change over levels in expi cells as assessed by the 2^((−ΔΔCt))method. Jurkat is included as a positive control. D49, 70, and 71indicate three individual donors.

FIGS. 31A-31B PVRIG binding characteristics to HEK hPVRIG engineeredcell lines, HEK parental cells, CA46 cells, and Jurkat cells. HEK OEdenotes HEK hPVRIG cells, HEK par denotes HEK parental cells. For Jurkatand CA46 data, gMFIr indicates the fold difference in geometric MFI ofPVRIG antibody staining relative to their controls. Concentrationindicates that at which the gMFIr was calculated. Not reliable fitindicates antibody binding characteristics do meet appropriatemathematical fitting requirements. Some antibodies were not tested insome conditions due to poor binding characteristics, specificity, ormanufacturability.

FIGS. 32A-32B PVRIG binding characteristics to primary human PBMC, cynotransient over-expressing cells, and cyno primary PBMC. Expi cyno OEdenotes expi cells transiently transfected with cPVRIG, expi par denotesexpi parental cells. gMFIr indicates the fold difference in geometricMFI of PVRIG antibody staining relative to their controls. Concentrationindicates that at which the gMFIr was calculated. Some antibodies werenot tested in some conditions due to poor binding characteristics,specificity, or manufacturability as in FIGS. 31A-31B. Additionally,select antibodies were triaged for screening on cyno PBMC subsets basedon their ability to bind cPVRIG transient cells or functionality.Expression of PVRIG on CD4 T cells is similar to that described in thetable for CD8 T cells.

FIG. 33 PVRIG antibody specificity towards CA46 and Jurkat cells. Datashows absolute geometric MFI (gMFI) measurements by FACS as a functionof increasing antibody concentration. The solid black line withtriangles shows staining of CA46 cells with anti-human PVRIG antibody(CPA.7.021) and the solid black line with squares shows staining ofJurkat cells. OV-90 (broken line with upside down triangles) andNCI-H4411 (broken line with diamonds) are shown as negative controls.

FIGS. 34A-34D PVRIG antibody cross-reactivity towards cPVRIG transientcells. Data shows an example of an antibody that is a negative binder(a-b, CPA.7.021) and a positive binder (c-d, CPA.7.024) on cPVRIGtransient cells. Solid grey histograms indicate control antibody, openblack histograms indicate the antibody of interest. Cells were stainedwith each antibody at a concentration of 5 μg/ml.

FIG. 35 cPVRIG RNA was assessed in sorted cyno PBMC subsets by qPCR.Data shown is the average Ct values from three cyno donors as detectedby two primer sets directed at two distinct areas of the cPVRIG gene.

FIGS. 36A-36C cPVRIG protein was assessed on a) CD16+ lymphocytes (NKcells), b) CD14+CD56+ myeloid cells (monocytes), and c) CD3+ lymphocytes(T cells) by FACS. Data is shown as absolute geometric MFI, with thesolid black line indicating background fluorescence levels. Data isrepresentative of a sample of our panel of anti-human PVRIG antibodiestested in three cyno donors.

FIGS. 37A-37B shows the CDR sequences for Fabs that were determined tosuccessfully block interaction of the PVRIG with its counterpart PVRL2,as described in Example 5.

FIGS. 38A-38AA shows the amino acid sequences of the variable heavy andlight domains, the full length heavy and light chains, and the variableheavy and variable light CDRs for the enumerated human CPA anti-PVRIGsequences of the invention that both bind PVRIG and block binding ofPVRIG and PVLR2.

FIGS. 39A-39H depicts the amino acid sequences of the variable heavy andlight domains, the full length heavy and light chains, and the variableheavy and variable light CDRs for eight human CPA anti-PVRIG sequencesof the invention that bind PVRIG and but do not block binding of PVRIGand PVLR2.

FIGS. 40A-40D depicts the CDRs for all CPA anti-PVRIG antibody sequencesthat were generated that bind PVRIG, including those that do not blockbinding of PVRIG and PVLR2.

FIGS. 41A to 41DD depicts the variable heavy and light chains as well asthe vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2 and vlCDR3 sequences of eachof the enumerated CHA antibodies of the invention, CHA.7.502, CHA.7.503,CHA.7.506, CHA.7.508, CHA.7.510, CHA.7.512, CHA.7.514, CHA.7.516,CHA.7.518, CHA.7.520.1, CHA.7.520.2, CHA.7.522, CHA.7.524, CHA.7.526,CHA.7.527, CHA.7.528, CHA.7.530, CHA.7.534, CHA.7.535, CHA.7.537,CHA.7.538.1, CHA.7.538.2, CHA.7.543, CHA.7.544, CHA.7.545, CHA.7.546,CHA.7.547, CHA.7.548, CHA.7.549 and CHA.7.550 (these include thevariable heavy and light sequences from mouse sequences (fromHybridomas).

FIG. 42 depicts the binning results from Example 11. Not binned:CPA.7.029 and CPA.7.026 (no binding to the antigen).

FIG. 43 Binary matrix of pair-wise blocking (“0”, red box) orsandwiching (“1”, green box) of antigen for 35 anti-PVRIG mAbs. MAbslisted vertically on the left of the matrix are mAbs covalentlyimmobilized to the ProteOn array. MAbs listed horizontally across thetop of the matrix were analytes injected with pre-mixed antigen. CloneCPA.7.041 was studied only as an analyte. The black boxes outline fourepitope bins according to the vertical blocking patterns of the mAbs.

FIG. 44 Hierarchical clustering dendrogram of the vertical bindingpatterns of each mAb in the binary matrix in FIG. 43. There are fourbins of mAbs with identical epitope blocking patterns within each group.The only difference between bins 1 and 2 is mAbs in bin 1 block antigenbinding to clone CPA.7.039 while mAbs in bin 2 can sandwich the antigenwith CPA.7.039. Clone CPA.7.050 can sandwich the antigen with all otherclones.

FIGS. 45A-45JJ Sensorgrams indicating the antigen blocking pattern forCPA.7.036 with all other immobilized mAbs, which are representative datafor Bin #1. Each panel represents a different ProteOn chip array spothaving a different immobilized mAb. Blue responses are antigen-onlycontrols. Black responses are pre-mixed solutions of CPA.7.036 in molarexcess of antigen. Gray responses are mAb-only control injections.CPA.7.36 blocks antigen binding to all other mAbs except for CPA.7.050(JJ).

FIGS. 46A-46JJ Sensorgrams indicating the antigen blocking pattern forCPA.7.034 with all other immobilized mAbs, which are representative datafor Bin #2. Each panel represents a different ProteOn chip array spothaving a different immobilized mAb. Blue responses are antigen-onlycontrols. Black responses are pre-mixed solutions of CPA.7.34 in molarexcess of antigen. Gray responses are mAb-only control injections.CPA.7.34 blocks antigen binding to all other mAbs except for CPA.7.039(DD) and CPA.7.050 (JJ).

FIGS. 47A-47JJ Sensorgrams indicating the antigen blocking pattern forCPA.7.039 with all other immobilized mAbs. CPA.7.039 is the only mAb inBin #3. Each panel represents a different ProteOn chip array spot havinga different immobilized mAb. Blue responses are antigen-only controls.Black responses are pre-mixed solutions of CPA.7.039 in molar excess ofantigen. Gray responses are mAb-only control injections. Panels C, F, H,J, L, N, R, S, Z, EE, GG, HH, II, and JJ show sandwiching of theantigen.

FIGS. 48A-48JJ Sensorgrams indicating the antigen blocking pattern forCPA.7.050 with all other immobilized mAbs. CPA.7.050 is the only mAb inBin #4. Each panel represents a different ProteOn chip array spot havinga different immobilized mAb. Blue responses are antigen-only controls.Black responses are pre-mixed solutions of CPA.7.50 in molar excess ofantigen. Gray responses are mAb-only control injections. Only panel JJshows antigen blocking which is where CPA.7.050 was injected w/antigenover itself.

FIG. 49 show the results of the SPR experiments of Example 12.

FIGS. 50A-50Q SPR sensorgram data of multiple concentrations of antiPVRIG fabs in supernatant injected over captured human PVRIG fusionprotein (black lines). The red lines show the 1:1 global kinetic fit tomultiple concentrations of the fabs to estimate the ka and ka of theinteractions. Letters indicate the clone listed in Table 1, which alsolists the resulting rate constants and calculated K_(D)

FIGS. 51A-51C SPR sensorgrams for clones CPA.7.009 (A), CPA.7.003 (B),and CPA.7.014 (C) binding to captured human PVRIG fusion protein. Theseare examples where the sensorgrams showed complex, multi-phasic kineticsand therefore the rate constants could not be reliably estimated.

FIGS. 52A-52B shows the results of the blocking studies from “AdditionalValidation Study 4” in Example 5.

FIG. 53 shows that following allo-activation, the expression of PVRIGwas upregulated on CD4+ T cells as well as on CD8+ T cells and doublenegative gamma delta T cells. This upregulation was observed in PBMCs ofone out of two donors tested.

FIG. 54 shows the human cell lines tested in Example 1G.

FIG. 55 shows the mouse cell lines tested in Example 1G.

FIGS. 56A-56C. Transcript expression of human PVRIG in various Humancancer cell lines. Verification of the human transcript in several celllines was performed by qRT-PCR using TaqMan probe. Column diagramrepresents data observed using TaqMan probe Hs04189293_g1. Ct values aredetailed in the table. Analysis indicating high transcript in Jurkat,HUT78 and HL60, and lower levels in THP1 and RPMI8226 cell lines.

FIGS. 57A-57B Transcript expression of mouse PVRIG in various mouse celllines. Verification of the mouse transcript in several cell lines wasperformed by qRT-PCR using TaqMan probe. Column diagram represents dataobserved using TaqMan probe CC70L8H. Ct values are detailed in thetable. Analysis indicating high transcript in NIH/3T3, Renca, SaI/N andJ774A.1, and lower levels in CT26 and B104-1-1 cell lines.

FIG. 58 Endogenous expression of PVRIG protein was analyzed by WB withthe commercial anti-human PVRIG rabbit polyclonal antibody (Sigma, cat#HPA047497), using whole cell extracts of various cell lines. Extractsof HEK293 cells ectopically over-expressing human PVRIG (lane 2) orcells transfected with empty vector (lane 1), were used as positive andnegative controls, respectively.

FIG. 59 qRT-PCR analysis of human PVRIG transcript in Jurkat cell linetransfected with PVRIG siRNA. Jurkat human cancer cell line, transfectedwith human PVRIG siRNA or with scrambled siRNA were analyzed by qRT-PCRusing human PVRIG TaqMan probe #Hs04189293_g1, and was normalized withgeo-mean of two housekeeping genes indicated in table above. Ct valuesare detailed in the table. Standard deviation of technical triplicatesof the PCR reaction are indicated.

FIG. 60 Membrane expression of human PVRIG protein in Jurkat human cellline transfected with human PVRIG siRNA. Jurkat cells transfected withHuman PVRIG siRNA were stained with monoclonal anti-PVRIG Ab Inc,CPA.7.021 (left panel, green line) or with IgG2 isotype control antibody(left panel, blue line) and with Sigma Ab (right panel, red line) orwith IgG (right panel, blue line). Cells transfected with ScrambledsiRNA were stained with the same anti-PVRIG (orange) or isotype control(left panel red line for mAb staining; right panel green line for SigmaAb). Following cell washing, PE-Goat anti-mouse secondary conjugated Abwas added to Sigma Ab only.

FIG. 61 indicates the summary of the findings described in this report,highlighting the cell lines showing correlation between qPCR and FACS,confirmed by knock down, HSKG-housekeeping gene, +− Positive, NT-NotTested, X-negative, KD-knockdown.

FIG. 62 indicates the summary of the findings described in this report,highlighting the cell lines showing correlation between qPCR and FACS,confirmed by knock down. HSKG-housekeeping gene, +− Positive, NT-NotTested, X-negative, KD-knockdown.

FIGS. 63A-63D depicts the vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2 andvlCDR3 sequences of each of the enumerated CPA antibodies of theinvention, CPA.7.001 to CPA.7.050 are human sequences (from Phagedisplay).

FIGS. 64A-64B shows the results of the screening in Example 1B.

FIG. 65 Antibodies specifics and staining concentration used in Example1I.

FIGS. 66A-66C depicts the sequences of human IgG1, IgG2, IgG3 and IgG4.

FIG. 67 depicts a number of human PVRIG ECD fragments.

FIG. 68 depicts the binding curve for CPA.7.021 as shown in EXAMPLE 13.

FIGS. 69A-69C Detection of CD137 and PD-1 surface expression. CD8+ Tcells, CD4+ T cells and TILs were activated and monitored over time at 4time-points as described in M&M. Resting or activated cells were firstgated for lymphocytes (FSC-A vs. SSC-A), followed by live cells gate,further gated for singlets (FSC-H vs. FSC-A), CD4/CD8 positive cells andfurther gated for CD137 and PD1. Surface expression of PD-1 (left) andCD137 (right) on (A) CD8+ T cells (B) CD4+ T cells and (C) TILs atdifferent time-points normalized to isotype control over the time courseof activation.

FIGS. 70A-70C PVRIG expression on resting and activated CD4+T and CD8+ Tcells. CD4+ and CD8+ T cells were activated and monitored over time at 4time-points as described in M&M. Cells were stained with viability dye,then incubated with anti-PVRIG and isotype control (7.5 μg/ml), andevaluated by flow cytometry. (A) Expression on CD4+ T cells. Expressionof PVRIG on live resting (time 0) and activated CD4+ cells followingsinglet gating for 24, 48, 72h and 144h compared to isotype control. (B)Expression on CD8+ T cells. Expression of PVRIG on live resting (time 0)and activated CD8+ cells following singlet gating for 24, 48, 72h and144h compared to isotype control. Shown are the Geometric Mean of thefluorescent intensity values obtained. (C) Normalization of foldinduction staining with anti-PVRIG-CPA.7.021 ab compared to human IgG2isotype over the time course of activation.

FIGS. 71A-71C PVRIG expression on resting and activated TILs. TILs Mart1and 209 were activated and monitored over time at 4 time-points asdescribed in M&M. Cells were stained with viability dye, then incubatedwith anti-PVRIG and isotype control (7.5 μg/ml), and evaluated by flowcytometry. (A) Expression on TIL Mart1. Expression of PVRIG on liveresting (time 0) and activated TIL following singlet gating for 24, 48,72h and 144h compared to isotype control. (B) Expression on TIL 209.Expression of PVRIG on live resting (time 0) and activated TIL followingsinglet gating for 24, 48, 72h and 144h compared to isotype control.Shown are the Geometric Mean of the fluorescent intensity valuesobtained. (C) Normalization of fold induction staining with antiPVRIG-CPA.7.021 ab compared with human IgG2 isotype control over thetime course of activation.

FIG. 72 Expression of PVRL2 on monocyte-derived DC. PVRL2 expression(triangles with broken line) as a function of time (days) relative toisotype control (circles with solid line) is shown. Day afterdifferentiation indicates time after addition of GM-CSF and IL-4 tomonocytes.

FIGS. 73A-73B Expression of PVRIG on CD4 and CD8 T cells in the MLR. Theexpression of PVRIG on proliferating (CFSE low) and non-proliferating Tcells (CFSE high) is shown. Data is derived from three individual CD3 Tcell donors and from a range of PVRIG antibodies. CFSE is measured onthe X axis and PVRIG expression is measured on the Y axis. The top 3series of scatter plots indicates PVRIG expression on CD4 T cells, andthe bottom 3 series indicates expression on CD8 T cells.

FIGS. 74A-74B Normalised expression of PVRIG on CD4 and CD8 T cells inthe MLR. The expression of PVRIG relative to mIgG1 isotype control isshown from three individual CD3 T cell donors across all antibodiesanalysed.

FIGS. 75A-75B PVRIG antibodies increase T cell proliferation in the MLR.The percentages of CFSE low cells are shown from MLR assays treated withthe indicated PVRIG antibodies. Each graph represents one individual CD3T cell donor.

FIG. 76 FACS-based epitope analysis of PVRIG antibodies on T cells. Thelevel of binding of conjugated CPA.7.021 (derived from phage campaign)is indicated after pre-incubation of T cells with unconjugated PVRIGantibodies derived from our hybridoma campaign, as well as relevantcontrols. Analysis was performed on CFSE low T cells derived from theMLR.

FIG. 77 PVRIG antibody specificity towards HEK cells engineered tooverexpress PVRIG. Data shows absolute geometric MFI (gMFI) measurementsas a function of increasing antibody concentration. The broken blackline with squares shows staining of HEK hPVRIG cells with arepresentative anti-human PVRIG antibody (CHA.7.518), and the solidblack line with circles shows staining of HEK parental cells with thesame antibody.

FIG. 78 PVRIG antibodies show specificity towards Jurkat cells. Datashows absolute geometric MFI (gMFI) measurements by FACS as a functionof increasing antibody concentration. The broken black line with squaresshows staining of Jurkat cells with anti-human PVRIG antibody(CHA.7.518) and the solid black line with circles shows staining with anmIgG1 control antibody.

FIGS. 79A-79B PVRIG hybridoma antibody binding characteristics to HEKhPVRIG engineered cell lines, HEK parental cells, and Jurkat cells. HEKOE denotes HEK hPVRIG cells, HEK par denotes HEK parental cells. ForJurkat data, gMFIr indicates the fold difference in geometric MFI ofPVRIG antibody staining relative to their controls. Concentrationindicates that at which the gMFIr was calculated. No binding indicatesantibody does not bind to the tested cell line. Highlighted antibodiesare the ‘top four’ antibodies of interest.

FIGS. 80A-80B PVRIG hybridoma antibody binding characteristics toprimary human PBMC, cyno over-expressing cells, and cyno primary PBMC.Expi cyno OE denotes expi cells transiently transfected with cPVRIG,expi par denotes expi parental cells. gMFIr indicates the folddifference in geometric MFI of PVRIG antibody staining relative to theircontrols. Concentrations indicate that at which the gMFIr wascalculated. Not tested indicates antibodies that were not tested due toan absence of binding to human HEK hPVRIG, expi cPVRIG cells, or notmeeting binding requirements to PBMC subsets. Highlighted antibodies arethe ‘top four’ antibodies of interest.

FIGS. 81A-81B Summary of blocking capacity of PVRIG antibodies in theFACS-based competition assay. The IC₅₀ of inhibition is indicated. NoIC₅₀ indicates that these antibodies are non-blockers. Highlightedantibodies are the ‘top four’ antibodies of interest.

FIG. 82 KD validation performed in TILs 24 hr post-electroporation withsiRNA. TILs were stained with anti PVRIG or anti PD-1 analyzed by FACS.Percentage of the KD population is calculated relative to SCR stainedwith the relevant Ab.

FIG. 83A-83C KD TILs (MART-1 specific) were co-cultured with melanomacells 624 in 1:1 E:T for 18 hr and stained with anti CD8a antibody aswell as anti CD137 antibody and analyzed by FACS. Geometric meanfluorescence intensity are plotted (A). Co-culture supernatant wascollected as well and tested in Th1 Th2 Th7 cytometric bead array assayto detect secreted cytokines. IFNγ and TNF levels were detected (B,C).The percentage effect of a treatment is calculated by comparing eachtreatment to SCR control. The figure shows representative data of 2independent experiments. Treatments were compared by Student's t-test(*P<0.05, **P<0.01) of triplicate samples.

FIGS. 84A-84B KD TILs (F4 gp100 specific) were co-cultured with melanomacells 624 in 1:3 E:T for 18 hr and stained with anti CD8a antibody aswell as anti CD137 antibody and analyzed by FACS. Geometric meanfluorescence intensity are plotted (A). Co-culture supernatant wascollected as well and tested in Th1 Th2 Th7 cytometric bead array assayto detect secreted cytokines. IFNγ levels were detected (B). Percentageof the effect a treatment has is calculated by comparing each treatmentto SCR control. Figure shows representative data of 2 independentexperiments. Treatments were compared by Student's t-test (*P<0.05,**P<0.01) of triplicate samples.

FIGS. 85A-85B TILs from were co-cultured with melanoma cells 624 at 1:1E:T for 18 hr in the presence of anti-PVRIG Ab (CPA.7.021; 10 μg/ml),anti-TIGIT (10A7 clone; 10 μg/ml) or in combination. Supernatant wascollected and tested in Th1 Th2 Th17 cytometric bead array assay todetect secreted cytokines. IFNγ (A) and TNF (B) levels were detected.Treatments were compared by Student's t-test (*P<0.05, **P<0.01) oftriplicate samples.

FIG. 86A-86F MART-1 or 209 TILs were co-cultured with melanoma cells 624at 1:1 E:T for 18 hr in the presence of anti-PVRIG Ab (CPA.7.021; 10μg/ml), anti-DNAM1 (DX11 clone; 10 μg/ml) or in in combination.Supernatant was collected and tested in Th1 Th2 Th17 cytometric beadarray assay to detect secreted cytokines. IFNγ (A,D) and TNF (B,E)levels were detected. TILs were stained for surface expression of CD137(C,F).

FIGS. 87A-87B TILs (F4) were co-cultured with melanoma cells 624 at 1:3E:T for 18 hr in the presence of anti-PVRIG Ab (CPA.7.021; 10 μg/ml),anti-TIGIT (10A7 clone; 10 μg/ml), anti-PD1 (mAb 1B8, Merck; 10 μg/ml)or in combination. Supernatant was collected and tested in Th1 Th2 Th17cytometric bead array assay to detect secreted cytokines. IFNγ (A) andTNF (B) levels were detected.

FIGS. 88A-88I I depict four humanized sequences for each of CHA.7.518,CHA.7.524, CHA.7.530, CHA.7.538_1 and CHA.7.538_2. Note that the lightchain for CHA.7.538_2 is the same as for CHA.7.538_1. The “H1” of eachis a “CDR swap” with no changes to the human framework. Subsequentsequences alter framework changes shown in larger bold font. CDRsequences are noted in bold. CDR definitions are AbM from websitewww.bioinf.org.uk/abs/. Human germline and joining sequences from IMGT®the international ImMunoGeneTics® information system www.imgt.org(founder and director: Marie-Paule Lefranc, Montpellier, France).Residue numbering shown as sequential (seq) or according to Chothia fromwebsite www.bioinf.org.uk/abs/(AbM). “b” notes buried sidechain; “p”notes partially buried; “i” notes sidechain at interface between VH andVL domains. Sequence differences between human and murine germlinesnoted by asterisk (*). Potential additional mutations in frameworks arenoted below sequence. Potential changes in CDR sequences noted beloweach CDR sequence as noted on the figure (# deamidation substitutions:Q/S/A; these may prevent asparagine (N) deamidation. @ tryptophanoxidation substitutions: Y/F/H; these may prevent tryptophan oxidation;@ methionine oxidation substitutions: L/F/A).

FIGS. 89A-89E depicts a collation of the humanized sequences of five CHAantibodies.

FIG. 90 depicts schemes for combining the humanized VH and VL CHAantibodies of FIGS. 88A-88I and FIGS. 89A-89E. The “chimVH” and “chimVL”are the mouse variable heavy and light sequences attached to a human IgGconstant domain.

FIG. 91 PVRIG hybridoma antibody binding characteristics to primaryhuman PBMC, cyno over-expressing cells, and cyno primary PBMC. Expi cynoOE denotes expi cells transiently transfected with cPVRIG, expi pardenotes expi parental cells. gMFIr indicates the fold difference ingeometric MFI of PVRIG antibody staining relative to their controls.Concentrations indicate that at which the gMFIr was calculated. Nottested indicates antibodies that were not tested due to an absence ofbinding to human HEK hPVRIG, expi cPVRIG cells, or not meeting bindingrequirements to PBMC subsets. Highlighted antibodies are four antibodiesfor which humanization was done (See FIG. 90).

FIG. 92 Summary of blocking capacity of PVRIG antibodies in theFACS-based competition assay. The IC50 of inhibition is indicated. NoIC50 indicates that these antibodies are non-blockers. Highlightedantibodies are four antibodies for which humanization was done (See FIG.90).

FIGS. 93A-93C Effect of PVRIG antibodies in blocking the interactionbetween PVRIG and PVRL2. (a-b) Data shows changes in absolute gMFIrepresenting changes in binding of soluble PVRIG to HEK cells when fourPVRIG antibodies are added to disrupt the interaction. Also indicatedare the IC₅₀ values of each antibody in each assay. A) Data showsdisruption of soluble PVRIG with HEK cells when the antibodies arepre-incubated with antigen. B) Data shows disruption of soluble PVRIGwith HEK cells when the antibodies are added concomitantly with antigen.C) Data shows changes in absolute gMFI representing changes in bindingof soluble PVRL2 Fc to HEK hPVRIG cells when four PVRIG antibodies areadded to disrupt the interaction. IC₅₀ values of each antibody areindicated. ND denotes not determined.

FIGS. 94A-94H NK cell receptor and ligand expression on Reh cells.Expression of NK cell receptors such as a) PVRIG, b) DNAM-1, c) TIGITare shown. Expression of NK receptor ligands such as d) PVR, e) PVRL2,f) ULBP2/5/6, g) ULBP3, and h) MICA/B are shown. Solid grey histogramsrepresent isotype controls and open black histograms represent theantibody of interest.

FIGS. 95A-95F Effect of PVRIG antibodies on enhancing NK cell-mediatedcytotoxicity against Reh cells. The effect of 5 μg/ml CPA.7.002 (a),CPA.7.005 (b), CPA.7.021 (a-c), and CPA.7.050 (c) was examined in NKcell cytotoxicity assays against Reh cells where the number of NK cellswas titrated against a constant number of Reh cells. d) The effect ofvarying the concentration of CPA.7.002 and CPA.7.021 on NK cell-mediatedcytotoxicity with a constant number of NK to Reh cells (5:1) wasexamined. DNAM-1 (e) and TIGIT (f) were examined in assays withconditions as outlined in panels a-c.

FIGS. 96A-96H NK cell receptor and ligand expression on MOLM-13 cells.Expression of NK cell receptors such as a) PVRIG, b) DNAM-1, c) TIGITare shown. Expression of NK receptor ligands such as d) PVR, e) PVRL2,f) ULBP2/5/6, g) ULBP3, and h) MICA/B are shown. Solid grey histogramsrepresent isotype controls and open black histograms represent theantibody of interest.

FIGS. 97A-97B Effect of PVRIG antibodies on enhancing NK cell-mediatedcytotoxicity against MOLM-13 cells. a) The effect of 5 μg/ml CPA.7.002,CPA.7.005, and CPA.7.021 was examined in NK cell cytotoxicity assaysagainst MOLM-13 cells where the number of NK cells was titrated againsta constant number of MOLM-13 cells. b) TIGIT was examined similar topanel a.

FIG. 98 Summary of blocking capacity of PVRIG antibodies in the cellularbiochemical assay. Assay permutation and orientation, and the IC₅₀ ofinhibition are indicated. (P) indicates the assay permutation wherePVRIG antibodies are pre-incubated with PVRIG antigen prior to additionto HEK cells. (NP) indicates the concomitant addition of PVRIGantibodies and PVRIG antigen to HEK cells. Increased binding indicatesthat PVRL2 Fc binding to HEK hPVRIG cells was enhanced, rather thaninhibited.

FIG. 99: Summary of the activity of select PVRIG antibodies in NK cellcytotoxicity assays against Reh and MOLM-13 cells. Fold change incytotoxicity relative to control was calculated by dividing the absolutelevel of killing (%) in the condition with PVRIG antibody, by theabsolute level of killing (%) with control antibody. Fold change iscalculated from the 5:1 effector to target ratio.

FIG. 100 Sequence alignment of PVRIG orthologs. Aligned sequences of thehuman, cynomolgus, marmoset, and rhesus PVRIG extra-cellular domain. Thedifferences between human and cynomolgus are highlighted in yellow.

FIG. 101 Binding of anti human PVRIG antibodies to cyno, human,cyno/human hybrid PVRIG variants. Binding of antibodies to wild typecyno PVRIG (●), H61R cyno PVRIG (▪), P67S cyno PVRIG (▴), L95R/T971 cynoPVRIG (▾), and wild type human PVRIG (♦) are shown. The ELISA signalsare plotted as a function of antibody concentration.

FIG. 102 Correlation of epitope group and cyno cross-reactivity ofanti-human PVRIG antibodies.

FIGS. 103A-103BX shows a number of sequences of use in the invention.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

Cancer can be considered as an inability of the patient to recognize andeliminate cancerous cells. In many instances, these transformed (e.g.cancerous) cells counteract immunosurveillance. There are naturalcontrol mechanisms that limit T-cell activation in the body to preventunrestrained T-cell activity, which can be exploited by cancerous cellsto evade or suppress the immune response. Restoring the capacity ofimmune effector cells-especially T cells—to recognize and eliminatecancer is the goal of immunotherapy. The field of immuno-oncology,sometimes referred to as “immunotherapy” is rapidly evolving, withseveral recent approvals of T cell checkpoint inhibitory antibodies suchas Yervoy, Keytruda and Opdivo. These antibodies are generally referredto as “checkpoint inhibitors” because they block normally negativeregulators of T cell immunity. It is generally understood that a varietyof immunomodulatory signals, both costimulatory and coinhibitory, can beused to orchestrate an optimal antigen-specific immune response.Generally, these antibodies bind to checkpoint inhibitor proteins suchas CTLA-4 and PD-1, which under normal circumstances prevent or suppressactivation of cytotoxic T cells (CTLs). By inhibiting the checkpointprotein, for example through the use of antibodies that bind theseproteins, an increased T cell response against tumors can be achieved.That is, these cancer checkpoint proteins suppress the immune response;when the proteins are blocked, for example using antibodies to thecheckpoint protein, the immune system is activated, leading to immunestimulation, resulting in treatment of conditions such as cancer andinfectious disease.

The present invention is directed to the use of antibodies to humanPoliovirus Receptor Related Immunoglobulin Domain Containing Protein, or“PVRIG”, sometimes also referred to herein as “PV protein”. PVRIG isexpressed on the cell surface of NK and T-cells and shares severalsimilarities to other known immune checkpoints.

Computational algorithms were used to analyze the human genome in orderto identify novel immune checkpoints. Genes were identified that arepredicted to be cell surface proteins, have an Ig domain and areexpressed on immune cells within the tumor microenvironment,specifically on tumor infiltrating lymphocytes (TILs), which arepresumed to be receptors. Proteins that have a single IgV domain andhave an intracellular ITIM-like motif were identified, which suggeststhat they are acting as immune checkpoint and have an inhibitory effecton T cells and/or NK cells. Once identified computationally, variousvalidation experiments were done, including: expression studiesdemonstrating that PVRIG is expressed on lymphocytes and on lymphocyteswithin the tumor microenvironment and has an inhibitory effect on NK andT cells (demonstrated both with knockdown experiments and withantibodies directed at PVRIG). PVRL2 was identified/confirmed to be thecounterpart of PVRIG. Antibodies that bind to PVRIG were generated, andthen a subset of those were identified that both bind to PVRIG and blockthe interaction of PVRIG and PVLR2.

Accordingly, when PVRIG is bound by its ligand (PVRL2), an inhibitorysignal is elicited which acts to attenuate the immune response of NK andT-cells against a target cell (i.e. analogous to PD-1/PDL1). Blockingthe binding of PVRL2 to PVRIG shuts-off this inhibitory signal of PVRIGand as a result modulates the immune response of NK and T-cells.Utilizing an antibody against PVRIG that blocks binding to PVRL2 is atherapeutic approach that could enhance the killing of cancer cells byNK and T-cells. Blocking antibodies have been generated which bind PVRIGand block the binding of its ligand, PVRL2.

As shown in the Example section, the expression of PVRIG has beenpositively correlated to expression of PD-1, a known immune checkpointprotein. Additionally, introduction of PVRIG (as a extracellular domain(ECD) fusion protein) was shown to inhibit the activation of T cells,and thus the use of anti-PVRIG antibodies leads to T cell activation.Accordingly, anti-PVRIG antibodies can be used to treat conditions forwhich T cell or NK cell activation is desired such as cancer.

Functional effects of PVRIG blocking antibodies on NK and T-cells can beassessed in vitro (and in some cases in vivo, as described more fullybelow) by measuring changes in the following parameters: proliferation,cytokine release and cell-surface makers. For NK cells, increases incell proliferation, cytotoxicity (ability to kill target cells asmeasured by increases in CD107a, granzyme, and perform expression, or bydirectly measuring target cells killing), cytokine production (e.g.IFN-γ and TNF), and cell surface receptor expression (e.g. CD25) isindicative of immune modulation, e.g. enhanced killing of cancer cells.For T-cells, increases in proliferation, increases in expression of cellsurface markers of activation (e.g. CD25, CD69, CD137, and PD1),cytotoxicity (ability to kill target cells), and cytokine production(e.g. IL-2, IL-4, IL-6, IFNγ, TNF-α, IL-10, IL-17A) are indicative ofimmune modulation, e.g. enhanced killing of cancer cells.

Accordingly, the present invention provides antibodies, includingantigen binding domains, that bind to human PVRIG pps and methods ofactivating T cells and/or NK cells to treat diseases such as cancer andinfectious diseases, and other conditions where increased immuneactivity results in treatment.

II. PVRIG Proteins

The present invention provides antibodies that specifically bind toPVRIG proteins. “Protein” in this context is used interchangeably with“polypeptide”, and includes peptides as well. The present inventionprovides antibodies that specifically bind to PVRIG proteins. PVRIG is atransmembrane domain protein of 326 amino acids in length, with a signalpeptide (spanning from amino acid 1 to 40), an extracellular domain(spanning from amino acid 41 to 171), a transmembrane domain (spanningfrom amino acid 172 to 190) and a cytoplasmic domain (spanning fromamino acid 191 to 326). The full length human PVRIG protein is shown inFIG. 25. There are two methionines that can be start codons, but themature proteins are identical.

Accordingly, as used herein, the term “PVRIG” or “PVRIG protein” or“PVRIG polypeptide” may optionally include any such protein, orvariants, conjugates, or fragments thereof, including but not limited toknown or wild type PVRIG, as described herein, as well as any naturallyoccurring splice variants, amino acid variants or isoforms, and inparticular the ECD fragment of PVRIG. The term “soluble” form of PVRIGis also used interchangeably with the terms “soluble ectodomain (ECD)”or “ectodomain” or “extracellular domain (ECD) as well as “fragments ofPVRIG polypeptides”, which may refer broadly to one or more of thefollowing optional polypeptides:

The PVRIG proteins contain an immunoglobulin (Ig) domain within theextracellular domain, which is a PVR-like Ig fold domain. The PVR-likeIg fold domain may be responsible for functional counterpart binding, byanalogy to the other B7 family members. The PVR-like Ig fold domain ofthe extracellular domain includes one disulfide bond formed betweenintra domain cysteine residues, as is typical for this fold and may beimportant for structure-function. These cysteines are located atresidues 22 and 93 (or 94). In one embodiment, there is provided asoluble fragment of PVRIG that can be used in testing of PVRIGantibodies.

Included within the definition of PVRIG proteins are PVRIG ECDfragments. Optionally, the PVRIG ECD fragments refer also to any one ofthe polypeptide sequences listed in FIG. 67, which are reasonablyexpected to comprise functional regions of the PVRIG protein. Thisexpectation is based on a systematic analysis of a set of proteincomplexes with solved 3D structures, which contained complexes of Igproteins (for example PDB ID 1i85 which describe the complex of CTLA4AND CD86). The intermolecular contact residues from each “co-structure”from each PDB were collected and projected on the sequence of PVRIG.Several regions with clusters of interacting residues supported byseveral contact maps were identified and synthesized as a series ofpeptides and are reasonably expected to mimic the structure of theintact full length protein and thereby modulate one or more of theeffects of PVRIG on immunity and on specific immune cell types.According to at least some embodiments of the invention, the PVRIG ECDfragments represented by polypeptide sequences listed in FIG. 67, arelocated as follows (as compared to human PVRIG ECD of FIG. 25, countingfrom the first amino acid of the ECD): PVRIG Fragment A is located atpositions 46 to 66; PVRIG Fragment B is located at positions 46 to 79;PVRIG Fragment C is located at positions 63 to 79; PVRIG Fragment D islocated at positions 91 to 106; PVRIG Fragment E is located at positions91 to 114; PVRIG Fragment F is located at positions 11 to 25; PVRIGFragment G is located at positions 3 to 24; PVRIG Fragment H is locatedat positions 18 to 36; PVRIG Fragment I is located at positions 29 to52; PVRIG Fragment J is located at positions 73-98.

As noted herein and more fully described below, anti-PVRIG antibodies(including antigen-binding fragments) that both bind to PVRIG andprevent activation by PVRL2 (e.g. most commonly by blocking theinteraction of PVRIG and PVLR2), are used to enhance T cell and/or NKcell activation and be used in treating diseases such as cancer andpathogen infection.

III. Antibodies

Accordingly, the invention provides anti-PVRIG antibodies. PVRIG, alsocalled Poliovirus Receptor Related Immunoglobulin Domain ContainingProtein, Q6DKI7 or C7orf15, relates to amino acid and nucleic acidsequences shown in RefSeq accession identifier NP_076975, shown in FIG.25. The antibodies of the invention are specific for the PVRIGextracellular domain as more fully outlined herein.

As is discussed below, the term “antibody” is used generally. Antibodiesthat find use in the present invention can take on a number of formatsas described herein, including traditional antibodies as well asantibody derivatives, fragments and mimetics, described below. Ingeneral, the term “antibody” includes any polypeptide that includes atleast one antigen binding domain, as more fully described below.Antibodies may be polyclonal, monoclonal, xenogeneic, allogeneic,syngeneic, or modified forms thereof, as described herein, withmonoclonal antibodies finding particular use in many embodiments. Insome embodiments, antibodies of the invention bind specifically orsubstantially specifically to PVRIG molecules. The terms “monoclonalantibodies” and “monoclonal antibody composition”, as used herein, referto a population of antibody molecules that contain only one species ofan antigen-binding site capable of immunoreacting with a particularepitope of an antigen, whereas the term “polyclonal antibodies” and“polyclonal antibody composition” refer to a population of antibodymolecules that contain multiple species of antigen-binding sites capableof interacting with a particular antigen. A monoclonal antibodycomposition, typically displays a single binding affinity for aparticular antigen with which it immunoreacts.

Traditional full length antibody structural units typically comprise atetramer. Each tetramer is typically composed of two identical pairs ofpolypeptide chains, each pair having one “light” (typically having amolecular weight of about 25 kDa) and one “heavy” chain (typicallyhaving a molecular weight of about 50-70 kDa). Human light chains areclassified as kappa and lambda light chains. The present invention isdirected to the IgG class, which has several subclasses, including, butnot limited to IgG1, IgG2, IgG3, and IgG4. Thus, “isotype” as usedherein is meant any of the subclasses of immunoglobulins defined by thechemical and antigenic characteristics of their constant regions. Whilethe exemplary antibodies herein designated “CPA” are based on IgG1 heavyconstant regions, as shown in FIGS. 38A-38AA, the anti-PVRIG antibodiesof the invention include those using IgG2, IgG3 and IgG4 sequences, orcombinations thereof. For example, as is known in the art, different IgGisotypes have different effector functions which may or may not bedesirable. Accordingly, the CPA antibodies of the invention can alsoswap out the IgG1 constant domains for IgG2, IgG3 or IgG4 constantdomains (depicted in FIGS. 66A-66C), with IgG2 and IgG4 findingparticular use in a number of situations, for example for ease ofmanufacture or when reduced effector function is desired, the latterbeing desired in some situations.

For the enumerated antibodies of the CHA designation, these are murineantibodies generated in hybridomas (the “H” designation), and thus ingeneral they are humanized as is known in the art, generally in theframework regions (F1 to F4 for each of the heavy and light variableregions), and then grafted onto human IgG1, IgG2, IgG3 or IgG4 constantheavy and light domains (depicted in FIGS. 66A-66C), again with IgG4finding particular use, as is more fully described below.

The amino-terminal portion of each chain includes a variable region ofabout 100 to 110 or more amino acids primarily responsible for antigenrecognition, generally referred to in the art and herein as the “Fvdomain” or “Fv region”. In the variable region, three loops are gatheredfor each of the V domains of the heavy chain and light chain to form anantigen-binding site. Each of the loops is referred to as acomplementarity-determining region (hereinafter referred to as a “CDR”),in which the variation in the amino acid sequence is most significant.“Variable” refers to the fact that certain segments of the variableregion differ extensively in sequence among antibodies. Variabilitywithin the variable region is not evenly distributed. Instead, the Vregions consist of relatively invariant stretches called frameworkregions (FRs) of 15-30 amino acids separated by shorter regions ofextreme variability called “hypervariable regions”.

Each VH and VL is composed of three hypervariable regions(“complementary determining regions,” “CDRs”) and four FRs, arrangedfrom amino-terminus to carboxy-terminus in the following order:FR-CDR1-FR2-CDR2-FR3-CDR3-FR4.

The hypervariable region generally encompasses amino acid residues fromabout amino acid residues 24-34 (LCDR1; “L” denotes light chain), 50-56(LCDR2) and 89-97 (LCDR3) in the light chain variable region and aroundabout 31-35B (HCDR1; “H” denotes heavy chain), 50-65 (HCDR2), and 95-102(HCDR3) in the heavy chain variable region, although sometimes thenumbering is shifted slightly as will be appreciated by those in theart; Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5 thEd. Public Health Service, National Institutes of Health, Bethesda, Md.(1991) and/or those residues forming a hypervariable loop (e.g. residues26-32 (LCDR1), 50-52 (LCDR2) and 91-96 (LCDR3) in the light chainvariable region and 26-32 (HCDR1), 53-55 (HCDR2) and 96-101 (HCDR3) inthe heavy chain variable region; Chothia and Lesk (1987) J. Mol. Biol.196:901-917. Specific CDRs of the invention are described below andshown in FIGS. 40A-40D.

The carboxy-terminal portion of each chain defines a constant regionprimarily responsible for effector function. Kabat et al. collectednumerous primary sequences of the variable regions of heavy chains andlight chains. Based on the degree of conservation of the sequences, theyclassified individual primary sequences into the CDR and the frameworkand made a list thereof (see SEQUENCES OF IMMUNOLOGICAL INTEREST, 5 thedition, NIH publication, No. 91-3242, E. A. Kabat et al., entirelyincorporated by reference).

In the IgG subclass of immunoglobulins, there are several immunoglobulindomains in the heavy chain. By “immunoglobulin (Ig) domain” herein ismeant a region of an immunoglobulin having a distinct tertiarystructure. Of interest in the present invention are the heavy chaindomains, including, the constant heavy (CH) domains and the hingedomains. In the context of IgG antibodies, the IgG isotypes each havethree CH regions. Accordingly, “CH” domains in the context of IgG are asfollows: “CH1” refers to positions 118-220 according to the EU index asin Kabat. “CH2” refers to positions 237-340 according to the EU index asin Kabat, and “CH3” refers to positions 341-447 according to the EUindex as in Kabat.

Accordingly, the invention provides variable heavy domains, variablelight domains, heavy constant domains, light constant domains and Fcdomains to be used as outlined herein. By “variable region” as usedherein is meant the region of an immunoglobulin that comprises one ormore Ig domains substantially encoded by any of the Vκ or Vλ, and/or VHgenes that make up the kappa, lambda, and heavy chain immunoglobulingenetic loci respectively. Accordingly, the variable heavy domaincomprises vhFR1-vhCDR1-vhFR2-vhCDR2-vhFR3-vhCDR3-vhFR4, and the variablelight domain comprises vlFR1-vlCDR1-vlFR2-vlCDR2-vlFR3-vlCDR3-vlFR4. By“heavy constant region” herein is meant the CH1-hinge-CH2-CH3 portion ofan antibody. By “Fc” or “Fc region” or “Fc domain” as used herein ismeant the polypeptide comprising the constant region of an antibodyexcluding the first constant region immunoglobulin domain and in somecases, part of the hinge. Thus Fc refers to the last two constant regionimmunoglobulin domains of IgA, IgD, and IgG, the last three constantregion immunoglobulin domains of IgE and IgM, and the flexible hingeN-terminal to these domains. For IgA and IgM, Fc may include the Jchain. For IgG, the Fc domain comprises immunoglobulin domains Cy2 andCy3 (Cy2 and Cy3) and the lower hinge region between Cy1 (Cy1) and Cy2(Cy2). Although the boundaries of the Fc region may vary, the human IgGheavy chain Fc region is usually defined to include residues C226 orP230 to its carboxyl-terminus, wherein the numbering is according to theEU index as in Kabat. In some embodiments, as is more fully describedbelow, amino acid modifications are made to the Fc region, for exampleto alter binding to one or more FcγR receptors or to the FcRn receptor.

Thus, “Fc variant” or “variant Fc” as used herein is meant a proteincomprising an amino acid modification in an Fc domain. The Fc variantsof the present invention are defined according to the amino acidmodifications that compose them. Thus, for example, N434S or 434S is anFc variant with the substitution serine at position 434 relative to theparent Fc polypeptide, wherein the numbering is according to the EUindex. Likewise, M428L/N434S defines an Fc variant with thesubstitutions M428L and N434S relative to the parent Fc polypeptide. Theidentity of the WT amino acid may be unspecified, in which case theaforementioned variant is referred to as 428L/434S. It is noted that theorder in which substitutions are provided is arbitrary, that is to saythat, for example, 428L/434S is the same Fc variant as M428L/N434S, andso on. For all positions discussed in the present invention that relateto antibodies, unless otherwise noted, amino acid position numbering isaccording to the EU index.

By “Fab” or “Fab region” as used herein is meant the polypeptide thatcomprises the VH, CH1, VL, and CL immunoglobulin domains. Fab may referto this region in isolation, or this region in the context of a fulllength antibody, antibody fragment or Fab fusion protein. By “Fv” or “Fvfragment” or “Fv region” as used herein is meant a polypeptide thatcomprises the VL and VH domains of a single antibody. As will beappreciated by those in the art, these generally are made up of twochains.

Throughout the present specification, either the IMTG numbering systemor the Kabat numbering system is generally used when referring to aresidue in the variable domain (approximately, residues 1-107 of thelight chain variable region and residues 1-113 of the heavy chainvariable region) (e.g, Kabat et al., supra (1991)). EU numbering as inKabat is generally used for constant domains and/or the Fc domains.

The CDRs contribute to the formation of the antigen-binding, or morespecifically, epitope binding site of antibodies. “Epitope” refers to adeterminant that interacts with a specific antigen binding site in thevariable region of an antibody molecule known as a paratope. Epitopesare groupings of molecules such as amino acids or sugar side chains andusually have specific structural characteristics, as well as specificcharge characteristics. A single antigen may have more than one epitope.

The epitope may comprise amino acid residues directly involved in thebinding (also called immunodominant component of the epitope) and otheramino acid residues, which are not directly involved in the binding,such as amino acid residues which are effectively blocked by thespecifically antigen binding peptide; in other words, the amino acidresidue is within the footprint of the specifically antigen bindingpeptide.

Epitopes may be either conformational or linear. A conformationalepitope is produced by spatially juxtaposed amino acids from differentsegments of the linear polypeptide chain. A linear epitope is oneproduced by adjacent amino acid residues in a polypeptide chain.Conformational and nonconformational epitopes may be distinguished inthat the binding to the former but not the latter is lost in thepresence of denaturing solvents.

An epitope typically includes at least 3, and more usually, at least 5or 8-10 amino acids in a unique spatial conformation. Antibodies thatrecognize the same epitope can be verified in a simple immunoassayshowing the ability of one antibody to block the binding of anotherantibody to a target antigen, for example “binning”. Specific bins aredescribed below.

Included within the definition of “antibody” is an “antigen-bindingportion” of an antibody (also used interchangeably with “antigen-bindingfragment”, “antibody fragment” and “antibody derivative”). That is, forthe purposes of the invention, an antibody of the invention has aminimum functional requirement that it bind to a PVRIG antigen. As willbe appreciated by those in the art, there are a large number of antigenfragments and derivatives that retain the ability to bind an antigen andyet have alternative structures, including, but not limited to, (i) theFab fragment consisting of VL, VH, CL and CH1 domains, (ii) the Fdfragment consisting of the VH and CH1 domains, (iii) F(ab′)2 fragments,a bivalent fragment comprising two linked Fab fragments (vii) singlechain Fv molecules (scFv), wherein a VH domain and a VL domain arelinked by a peptide linker which allows the two domains to associate toform an antigen binding site (Bird et al., 1988, Science 242:423-426,Huston et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:5879-5883,entirely incorporated by reference), (iv) “diabodies” or “triabodies”,multivalent or multispecific fragments constructed by gene fusion(Tomlinson et. al., 2000, Methods Enzymol. 326:461-479; WO94/13804;Holliger et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90:6444-6448, allentirely incorporated by reference), (v) “domain antibodies” or “dAb”(sometimes referred to as an “immunoglobulin single variable domain”,including single antibody variable domains from other species such asrodent (for example, as disclosed in WO 00/29004), nurse shark andCamelid V-HH dAbs, (vi) SMIPs (small molecule immunopharmaceuticals),camelbodies, nanobodies and IgNAR.

Still further, an antibody or antigen-binding portion thereof(antigen-binding fragment, antibody fragment, antibody portion) may bepart of a larger immunoadhesion molecules (sometimes also referred to as“fusion proteins”), formed by covalent or noncovalent association of theantibody or antibody portion with one or more other proteins orpeptides. Examples of immunoadhesion molecules include use of thestreptavidin core region to make a tetrameric scFv molecule and use of acysteine residue, a marker peptide and a C-terminal polyhistidine tag tomake bivalent and biotinylated scFv molecules. Antibody portions, suchas Fab and F(ab′)2 fragments, can be prepared from whole antibodiesusing conventional techniques, such as papain or pepsin digestion,respectively, of whole antibodies. Moreover, antibodies, antibodyportions and immunoadhesion molecules can be obtained using standardrecombinant DNA techniques, as described herein.

In general, the anti-PVRIG antibodies of the invention are recombinant.“Recombinant” as used herein, refers broadly with reference to aproduct, e.g., to a cell, or nucleic acid, protein, or vector, indicatesthat the cell, nucleic acid, protein or vector, has been modified by theintroduction of a heterologous nucleic acid or protein or the alterationof a native nucleic acid or protein, or that the cell is derived from acell so modified. Thus, for example, recombinant cells express genesthat are not found within the native (non-recombinant) form of the cellor express native genes that are otherwise abnormally expressed, underexpressed or not expressed at all.

The term “recombinant antibody”, as used herein, includes all antibodiesthat are prepared, expressed, created or isolated by recombinant means,such as (a) antibodies isolated from an animal (e.g., a mouse) that istransgenic or transchromosomal for human immunoglobulin genes or ahybridoma prepared therefrom (described further below), (b) antibodiesisolated from a host cell transformed to express the human antibody,e.g., from a transfectoma, (c) antibodies isolated from a recombinant,combinatorial human antibody library, and (d) antibodies prepared,expressed, created or isolated by any other means that involve splicingof human immunoglobulin gene sequences to other DNA sequences. Suchrecombinant human antibodies have variable regions in which theframework and CDR regions are derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies can be subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the VH and VL regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline VH and VL sequences, may not naturally existwithin the human antibody germline repertoire in vivo.

A. Optional Antibody Engineering

The antibodies of the invention can be modified, or engineered, to alterthe amino acid sequences by amino acid substitutions.

By “amino acid substitution” or “substitution” herein is meant thereplacement of an amino acid at a particular position in a parentpolypeptide sequence with a different amino acid. In particular, in someembodiments, the substitution is to an amino acid that is not naturallyoccurring at the particular position, either not naturally occurringwithin the organism or in any organism. For example, the substitutionE272Y refers to a variant polypeptide, in this case an Fc variant, inwhich the glutamic acid at position 272 is replaced with tyrosine. Forclarity, a protein which has been engineered to change the nucleic acidcoding sequence but not change the starting amino acid (for exampleexchanging CGG (encoding arginine) to CGA (still encoding arginine) toincrease host organism expression levels) is not an “amino acidsubstitution”; that is, despite the creation of a new gene encoding thesame protein, if the protein has the same amino acid at the particularposition that it started with, it is not an amino acid substitution.

As discussed herein, amino acid substitutions can be made to alter theaffinity of the CDRs for the PVRIG protein (including both increasingand decreasing binding, as is more fully outlined below), as well as toalter additional functional properties of the antibodies. For example,the antibodies may be engineered to include modifications within the Fcregion, typically to alter one or more functional properties of theantibody, such as serum half-life, complement fixation, Fc receptorbinding, and/or antigen-dependent cellular cytotoxicity. Furthermore, anantibody according to at least some embodiments of the invention may bechemically modified (e.g., one or more chemical moieties can be attachedto the antibody) or be modified to alter its glycosylation, again toalter one or more functional properties of the antibody. Suchembodiments are described further below. The numbering of residues inthe Fc region is that of the EU index of Kabat.

In one embodiment, the hinge region of C_(H1) is modified such that thenumber of cysteine residues in the hinge region is altered, e.g.,increased or decreased. This approach is described further in U.S. Pat.No. 5,677,425 by Bodmer et al. The number of cysteine residues in thehinge region of CH1 is altered to, for example, facilitate assembly ofthe light and heavy chains or to increase or decrease the stability ofthe antibody.

In another embodiment, the Fc hinge region of an antibody is mutated todecrease the biological half-life of the antibody. More specifically,one or more amino acid mutations are introduced into the CH2-CH3 domaininterface region of the Fc-hinge fragment such that the antibody hasimpaired Staphylococcyl protein A (SpA) binding relative to nativeFc-hinge domain SpA binding. This approach is described in furtherdetail in U.S. Pat. No. 6,165,745 by Ward et al.

In some embodiments, amino acid substitutions can be made in the Fcregion, in general for altering binding to FcγR receptors. By “Fc gammareceptor”, “FcγR” or “FcgammaR” as used herein is meant any member ofthe family of proteins that bind the IgG antibody Fc region and isencoded by an FcγR gene. In humans this family includes but is notlimited to FcγRI (CD64), including isoforms FcγRIa, FcγRIb, and FcγRIc;FcγRII (CD32), including isoforms FcγRIIa (including allotypes H131 andR131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; andFcγRIII (CD16), including isoforms FcγRIIIa (including allotypes V158and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 andFcγRIIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, entirelyincorporated by reference), as well as any undiscovered human FcγRs orFcγR isoforms or allotypes. An FcγR may be from any organism, includingbut not limited to humans, mice, rats, rabbits, and monkeys. Mouse FcγRsinclude but are not limited to FcγRI (CD64), FcγRII (CD32), FcγRIII-1(CD16), and FcγRIII-2 (CD16-2), as well as any undiscovered mouse FcγRsor FcγR isoforms or allotypes.

There are a number of useful Fc substitutions that can be made to alterbinding to one or more of the FcγR receptors. Substitutions that resultin increased binding as well as decreased binding can be useful. Forexample, it is known that increased binding to FcγRIIIa generallyresults in increased ADCC (antibody dependent cell-mediatedcytotoxicity; the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause lysis of the target cell. Similarly, decreasedbinding to FcγRIIb (an inhibitory receptor) can be beneficial as well insome circumstances. Amino acid substitutions that find use in thepresent invention include those listed in U.S. Ser. Nos. 11/124,620(particularly FIG. 41) and U.S. Pat. No. 6,737,056, both of which areexpressly incorporated herein by reference in their entirety andspecifically for the variants disclosed therein. Particular variantsthat find use include, but are not limited to, 236A, 239D, 239E, 332E,332D, 239D/332E, 267D, 267E, 328F, 267E/328F, 236A/332E, 239D/332E/330Y,239D, 332E/330L, 299T and 297N.

In addition, the antibodies of the invention are modified to increaseits biological half-life. Various approaches are possible. For example,one or more of the following mutations can be introduced: T252L, T254S,T256F, as described in U.S. Pat. No. 6,277,375 to Ward. Alternatively,to increase the biological half-life, the antibody can be altered withinthe C_(H1) or C_(L) region to contain a salvage receptor binding epitopetaken from two loops of a CH2 domain of an Fc region of an IgG, asdescribed in U.S. Pat. Nos. 5,869,046 and 6,121,022 by Presta et al.Additional mutations to increase serum half life are disclosed in U.S.Pat. Nos. 8,883,973, 6,737,056 and 7,371,826, and include 428L, 434A,434S, and 428L/434S.

In yet other embodiments, the Fc region is altered by replacing at leastone amino acid residue with a different amino acid residue to alter theeffector functions of the antibody. For example, one or more amino acidsselected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and322 can be replaced with a different amino acid residue such that theantibody has an altered affinity for an effector ligand but retains theantigen-binding ability of the parent antibody. The effector ligand towhich affinity is altered can be, for example, an Fc receptor or the C1component of complement. This approach is described in further detail inU.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.

In another example, one or more amino acids selected from amino acidresidues 329, 331 and 322 can be replaced with a different amino acidresidue such that the antibody has altered C1q binding and/or reduced orabolished complement dependent cytotoxicity (CDC). This approach isdescribed in further detail in U.S. Pat. No. 6,194,551 by Idusogie etal.

In another example, one or more amino acid residues within amino acidpositions 231 and 239 are altered to thereby alter the ability of theantibody to fix complement. This approach is described further in PCTPublication WO 94/29351 by Bodmer et al.

In yet another example, the Fc region is modified to increase theability of the antibody to mediate antibody dependent cellularcytotoxicity (ADCC) and/or to increase the affinity of the antibody foran Fcγ receptor by modifying one or more amino acids at the followingpositions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268,269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294,295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326,327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378,382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or439. Thisapproach is described further in PCT Publication WO 00/42072 by Presta.Moreover, the binding sites on human IgG1 for FcγRI, FcγRII, FcγRIII andFcRn have been mapped and variants with improved binding have beendescribed (see Shields, R. L. et al. (2001) J. Biol. Chem.276:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334and 339 are shown to improve binding to FcγRIII. Additionally, thefollowing combination mutants are shown to improve FcγRIII binding:T256A/S298A, S298A/E333A, S298A/K224A and S298A/E333A/K334A.Furthermore, mutations such as M252Y/S254T/T256E or M428L/N434S improvebinding to FcRn and increase antibody circulation half-life (see Chan CA and Carter P J (2010) Nature Rev Immunol 10:301-316).

In still another embodiment, the antibody can be modified to abrogate invivo Fab arm exchange. Specifically, this process involves the exchangeof IgG4 half-molecules (one heavy chain plus one light chain) betweenother IgG4 antibodies that effectively results in bispecific antibodieswhich are functionally monovalent. Mutations to the hinge region andconstant domains of the heavy chain can abrogate this exchange (seeAalberse, RC, Schuurman J., 2002, Immunology 105:9-19).

In still another embodiment, the glycosylation of an antibody ismodified. For example, an aglycosylated antibody can be made (i.e., theantibody lacks glycosylation). Glycosylation can be altered to, forexample, increase the affinity of the antibody for antigen or reduceeffector function such as ADCC. Such carbohydrate modifications can beaccomplished by, for example, altering one or more sites ofglycosylation within the antibody sequence, for example N297. Forexample, one or more amino acid substitutions can be made that result inelimination of one or more variable region framework glycosylation sitesto thereby eliminate glycosylation at that site.

Additionally or alternatively, an antibody can be made that has analtered type of glycosylation, such as a hypofucosylated antibody havingreduced amounts of fucosyl residues or an antibody having increasedbisecting GlcNac structures. Such altered glycosylation patterns havebeen demonstrated to increase the ADCC ability of antibodies. Suchcarbohydrate modifications can be accomplished by, for example,expressing the antibody in a host cell with altered glycosylationmachinery. Cells with altered glycosylation machinery have beendescribed in the art and can be used as host cells in which to expressrecombinant antibodies according to at least some embodiments of theinvention to thereby produce an antibody with altered glycosylation. Forexample, the cell lines Ms704, Ms705, and Ms709 lack thefucosyltransferase gene, FUT8 (α(1,6) fucosyltransferase), such thatantibodies expressed in the Ms704, Ms705, and Ms709 cell lines lackfucose on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8 celllines are created by the targeted disruption of the FUT8 gene inCHO/DG44 cells using two replacement vectors (see U.S. PatentPublication No. 20040110704 by Yamane et al. and Yamane-Ohnuki et al.(2004) Biotechnol Bioeng 87:614-22). As another example, EP 1,176,195 byHanai et al. describes a cell line with a functionally disrupted FUT8gene, which encodes a fucosyl transferase, such that antibodiesexpressed in such a cell line exhibit hypofucosylation by reducing oreliminating the α 1,6 bond-related enzyme. Hanai et al. also describecell lines which have a low enzyme activity for adding fucose to theN-acetylglucosamine that binds to the Fc region of the antibody or doesnot have the enzyme activity, for example the rat myeloma cell lineYB2/0 (ATCC CRL 1662). PCT Publication WO 03/035835 by Presta describesa variant CHO cell line, Lec13 cells, with reduced ability to attachfucose to Asn(297)-linked carbohydrates, also resulting inhypofucosylation of antibodies expressed in that host cell (see alsoShields, R. L. et al. (2002) J. Biol. Chem. 277:26733-26740). PCTPublication WO 99/54342 by Umana et al. describes cell lines engineeredto express glycoprotein-modifying glycosyl transferases (e.g.,β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such thatantibodies expressed in the engineered cell lines exhibit increasedbisecting GlcNac structures which results in increased ADCC activity ofthe antibodies (see also Umana et al. (1999) Nat. Biotech. 17:176-180).Alternatively, the fucose residues of the antibody may be cleaved offusing a fucosidase enzyme. For example, the fucosidase α-L-fucosidaseremoves fucosyl residues from antibodies (Tarentino, A. L. et al. (1975)Biochem. 14:5516-23).

Another modification of the antibodies herein that is contemplated bythe invention is pegylation or the addition of other water solublemoieties, typically polymers, e.g., in order to enhance half-life. Anantibody can be pegylated to, for example, increase the biological(e.g., serum) half-life of the antibody. To pegylate an antibody, theantibody, or fragment thereof, typically is reacted with polyethyleneglycol (PEG), such as a reactive ester or aldehyde derivative of PEG,under conditions in which one or more PEG groups become attached to theantibody or antibody fragment. Preferably, the pegylation is carried outvia an acylation reaction or an alkylation reaction with a reactive PEGmolecule (or an analogous reactive water-soluble polymer). As usedherein, the term “polyethylene glycol” is intended to encompass any ofthe forms of PEG that have been used to derivatize other proteins, suchas mono (C₁-C₁₀) alkoxy- or aryloxy-polyethylene glycol or polyethyleneglycol-maleimide. In certain embodiments, the antibody to be pegylatedis an aglycosylated antibody. Methods for pegylating proteins are knownin the art and can be applied to the antibodies according to at leastsome embodiments of the invention. See for example, EP 0 154 316 byNishimura et al. and EP 0 401 384 by Ishikawa et al.

In addition to substitutions made to alter binding affinity to FcγRsand/or FcRn and/or increase in vivo serum half life, additional antibodymodifications can be made, as described in further detail below.

In some cases, affinity maturation is done. Amino acid modifications inthe CDRs are sometimes referred to as “affinity maturation”. An“affinity matured” antibody is one having one or more alteration(s) inone or more CDRs which results in an improvement in the affinity of theantibody for antigen, compared to a parent antibody which does notpossess those alteration(s). In some cases, although rare, it may bedesirable to decrease the affinity of an antibody to its antigen, butthis is generally not preferred.

In some embodiments, one or more amino acid modifications are made inone or more of the CDRs of the VISG1 antibodies of the invention. Ingeneral, only 1 or 2 or 3-amino acids are substituted in any single CDR,and generally no more than from 1, 2, 3, 4, 5, 6, 7, 8 9 or 10 changesare made within a set of CDRs. However, it should be appreciated thatany combination of no substitutions, 1, 2 or 3 substitutions in any CDRcan be independently and optionally combined with any othersubstitution.

Affinity maturation can be done to increase the binding affinity of theantibody for the PVRIG antigen by at least about 10% to 50-100-150% ormore, or from 1 to 5 fold as compared to the “parent” antibody.Preferred affinity matured antibodies will have nanomolar or evenpicomolar affinities for the PVRIG antigen. Affinity matured antibodiesare produced by known procedures. See, for example, Marks et al., 1992,Biotechnology 10:779-783 that describes affinity maturation by variableheavy chain (VH) and variable light chain (VL) domain shuffling. Randommutagenesis of CDR and/or framework residues is described in: Barbas, etal. 1994, Proc. Nat. Acad. Sci, USA 91:3809-3813; Shier et al., 1995,Gene 169:147-155; Yelton et al., 1995, J. Immunol. 155:1994-2004;Jackson et al., 1995, J. Immunol. 154(7):3310-9; and Hawkins et al,1992, J. Mol. Biol. 226:889-896, for example.

Alternatively, amino acid modifications can be made in one or more ofthe CDRs of the antibodies of the invention that are “silent”, e.g. thatdo not significantly alter the affinity of the antibody for the antigen.These can be made for a number of reasons, including optimizingexpression (as can be done for the nucleic acids encoding the antibodiesof the invention).

Thus, included within the definition of the CDRs and antibodies of theinvention are variant CDRs and antibodies; that is, the antibodies ofthe invention can include amino acid modifications in one or more of theCDRs of the enumerated antibodies of the invention. In addition, asoutlined below, amino acid modifications can also independently andoptionally be made in any region outside the CDRs, including frameworkand constant regions.

IV. PVRIG Antibodies

The present invention provides anti-PVRIG antibodies. (For convenience,“anti-PVRIG antibodies” and “PVRIG antibodies” are usedinterchangeably). The anti-PVRIG antibodies of the inventionspecifically bind to human PVRIG, and preferably the ECD of human VISG1,as depicted in FIG. 25.

Specific binding for PVRIG or a PVRIG epitope can be exhibited, forexample, by an antibody having a KD of at least about 10⁻⁷ M, at leastabout 10⁻⁵ M, at least about 10⁻⁶ M, at least about 10⁻⁷ M, at leastabout 10⁻⁸ M, at least about 10⁻⁹ M, alternatively at least about 10⁻¹⁰M, at least about 10⁻¹¹ M, at least about 10⁻¹² M, or greater, where KDrefers to a dissociation rate of a particular antibody-antigeninteraction. Typically, an antibody that specifically binds an antigenwill have a KD that is 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- ormore times greater for a control molecule relative to the PVRIG antigenor epitope.

However, as shown in the Examples, for optimal binding to PVRIGexpressed on the surface of NK and T-cells, the antibodies preferablyhave a KD less 50 nM and most preferably less than 1 nM, with less than0.1 nM and less than 1 pM and 0.1 pM finding use in the methods of theinvention.

Also, specific binding for a particular antigen or an epitope can beexhibited, for example, by an antibody having a KA or Ka for a PVRIGantigen or epitope of at least 20-, 50-, 100-, 500-, 1000-, 5,000-,10,000- or more times greater for the epitope relative to a control,where KA or Ka refers to an association rate of a particularantibody-antigen interaction.

In some embodiments, the anti-PVRIG antibodies of the invention bind tohuman PVRIG with a KD of 100 nM or less, 50 nM or less, 10 nM or less,or 1 nM or less (that is, higher binding affinity), or 1 pM or less,wherein KD is determined by known methods, e.g. surface plasmonresonance (SPR, e.g. Biacore assays), ELISA, KINEXA, and most typicallySPR at 25° or 37° C.

A. Specific Anti-PVRIG Antibodies

The invention provides antigen binding domains, including full lengthantibodies, which contain a number of specific, enumerated sets of 6CDRs.

The antibodies described herein as labeled as follows. The antibodieshave reference numbers, for example “CPA.7.013”. This represents thecombination of the variable heavy and variable light chains, as depictedin FIGS. 38A-38AA and FIGS. 39A-39H for example. “CPA.7.013.VH” refersto the variable heavy portion of CPA.7.013, while “CPA.7.013.VL” is thevariable light chain. “CPA.7.013.vhCDR1”, “CPA.7.013.vhCDR2”,“CPA.7.013.vhCDR3”, “CPA.7.013.vlCDR1”, “CPA.7.013.vlCDR2”, and“CPA.7.013.vlCDR3”, refers to the CDRs are indicated. “CPA.7.013.HC”refers to the entire heavy chain (e.g. variable and constant domain) ofthis molecule, and “CPA.7.013.LC” refers to the entire light light chain(e.g. variable and constant domain) of the same molecule. “CPA.7.013.H1”refers to a full length antibody comprising the variable heavy and lightdomains, including the constant domain of Human IgG1 (hence, the H1;IgG1, IgG2, IgG3 and IgG4 sequences are shown in FIGS. 66A-66C).Accordingly, “CPA.7.013.H2” would be the CPA.7.013 variable domainslinked to a Human IgG2. “CPA.7.013.H3” would be the CPA.7.013 variabledomains linked to a Human IgG3, and “CPA.7.013.H4” would be theCPA.7.013 variable domains linked to a Human IgG4.

The invention further provides variable heavy and light domains as wellas full length heavy and light chains.

In many embodiments, the antibodies of the invention are human (derivedfrom phage) and block binding of PVRIG and PVLR2. As shown in FIGS.52A-52B, the CPA antibodies that both bind and block the receptor-ligandinteraction are as below, with their components outlined as well:

CPA.7.001, CPA.7.001.VH, CPA.7.001.VL, CPA.7.001.HC, CPA.7.001.LC andCPA.7.001.H1, CPA.7.001.H2, CPA.7.001.H3, CPA.7.001.H4;CPA.7.001.vhCDR1, CPA.7.001.vhCDR2, CPA.7.001.vhCDR3, CPA.7.001.vlCDR1,CPA.7.001.vlCDR2, and CPA.7.001.vlCDR3;

CPA.7.003, CPA.7.003.VH, CPA.7.003.VL, CPA.7.003.HC, CPA.7.003.LC,CPA.7.003.H1, CPA.7.003.H2, CPA.7.003.H3, CPA.7.003.H4;CPA.7.003.vhCDR1, CPA.7.003.vhCDR2, CPA.7.003.vhCDR3, CPA.7.003.vlCDR1,CPA.7.003.vlCDR2, and CPA.7.003.vlCDR3;

CPA.7.004, CPA.7.004.VH, CPA.7.004.VL, CPA.7.004.HC, CPA.7.004.LC,CPA.7.004.H1, CPA.7.004.H2, CPA.7.004.H3 CPA.7.004.H4; CPA.7.004.vhCDR1,CPA.7.004.vhCDR2, CPA.7.004.vhCDR3, CPA.7.004.vlCDR1, CPA.7.004.vlCDR2,and CPA.7.004.vlCDR3;

CPA.7.006, CPA.7.006.VH, CPA.7.006.VL, CPA.7.006.HC, CPA.7.006.LC,CPA.7.006.H1, CPA.7.006.H2, CPA.7.006.H3 CPA.7.006.H4; CPA.7.006.vhCDR1,CPA.7.006.vhCDR2, CPA.7.006.vhCDR3, CPA.7.006.vlCDR1, CPA.7.006.vlCDR2,and CPA.7.006.vlCDR3;

CPA.7.008, CPA.7.008.VH, CPA.7.008.VL, CPA.7.008.HC, CPA.7.008.LC,CPA.7.008.H1, CPA.7.008.H2, CPA.7.008.H3 CPA.7.008.H4; CPA.7.008.vhCDR1,CPA.7.008.vhCDR2, CPA.7.008.vhCDR3, CPA.7.008.vlCDR1, CPA.7.008.vlCDR2,and CPA.7.008.vlCDR3;

CPA.7.009, CPA.7.009.VH, CPA.7.009.VL, CPA.7.009.HC, CPA.7.009.LC,CPA.7.009.H1, CPA.7.009.H2, CPA.7.009.H3 CPA.7.009.H4; CPA.7.009.vhCDR1,CPA.7.009.vhCDR2, CPA.7.009.vhCDR3, CPA.7.009.vlCDR1, CPA.7.009.vlCDR2,and CPA.7.009.vlCDR3;

CPA.7.010, CPA.7.010.VH, CPA.7.010.VL, CPA.7.010.HC, CPA.7.010.LC,CPA.7.010.H1, CPA.7.010.H2, CPA.7.010.H3 CPA.7.010.H4; CPA.7.010.vhCDR1,CPA.7.010.vhCDR2, CPA.7.010.vhCDR3, CPA.7.010.v1CDR1, CPA.7.010.v1CDR2,and CPA.7.010.v1CDR3;

CPA.7.011, CPA.7.011.VH, CPA.7.011.VL, CPA.7.011.HC, CPA.7.011.LC,CPA.7.011.H1, CPA.7.011.H2, CPA.7.011.H3 CPA.7.011.H4; CPA.7.011.vhCDR1,CPA.7.011.vhCDR2, CPA.7.011.vhCDR3, CPA.7.011.v1CDR1, CPA.7.011.vlCDR2,and CPA.7.011.v1CDR3;

CPA.7.012, CPA.7.012.VH, CPA.7.012.VL, CPA.7.012.HC, CPA.7.012.LC,CPA.7.012.H1, CPA.7.012.H2, CPA.7.012.H3 CPA.7.012.H4; CPA.7.012.vhCDR1,CPA.7.012.vhCDR2, CPA.7.012.vhCDR3, CPA.7.012.v1CDR1, CPA.7.012.vCDR2,and CPA.7.012.v1CDR3;

CPA.7.013, CPA.7.013.VH, CPA.7.013.VL, CPA.7.013.HC, CPA.7.013.LC,CPA.7.013.H1, CPA.7.013.H2, CPA.7.013.H3 CPA.7.013.H4; CPA.7.013.vhCDR1,CPA.7.013.vhCDR2, CPA.7.013.vhCDR3, CPA.7.013.v1CDR1, CPA.7.013.vCDR2,and CPA.7.013.v1CDR3;

CPA.7.014, CPA.7.014.VH, CPA.7.014.VL, CPA.7.014.HC, CPA.7.014.LC,CPA.7.014.H1, CPA.7.014.H2, CPA.7.014.H3 CPA.7.014.H4; CPA.7.014.vhCDR1,CPA.7.014.vhCDR2, CPA.7.014.vhCDR3, CPA.7.014.v1CDR1, CPA.7.014.vCDR2,and CPA.7.014.v1CDR3;

CPA.7.015, CPA.7.015.VH, CPA.7.015.VL, CPA.7.015.HC, CPA.7.015.LC,CPA.7.015.H1, CPA.7.015.H2, CPA.7.015.H3 CPA.7.015.H4; CPA.7.015.vhCDR1,CPA.7.015.vhCDR2, CPA.7.015.vhCDR3, CPA.7.015.v1CDR1, CPA.7.015.vCDR2,and CPA.7.015.v1CDR3;

CPA.7.017, CPA.7.017.VH, CPA.7.017.VL, CPA.7.017.HC, CPA.7.017.LC,CPA.7.017H1, CPA.7.017.H2, CPA.7.017.H3 CPA.7.017.H4; CPA.7.017.vhCDR1,CPA.7.000171.vhCDR2, CPA.7.017.vhCDR3, CPA.7.017.v1CDR1,CPA.7.017.vCDR2, and CPA.7.017.v1CDR3;

CPA.7.018, CPA.7.018.VH, CPA.7.018.VL, CPA.7.018.HC, CPA.7.018.LC,CPA.7.018.H1, CPA.7.018.H2, CPA.7.018.H3 CPA.7.018.H4; CPA.7.017.vhCDR1,CPA.7.017.vhCDR2, CPA.7.017.vhCDR3, CPA.7.017.v1CDR1, CPA.7.017.v1CDR2,and CPA.7.017.v1CDR3;

CPA.7.019, CPA.7.019.VH, CPA.7.019.VL, CPA.7.019.HC, CPA.7.019.LC,CPA.7.019.H1, CPA.7.019.H2, CPA.7.019.H3 CPA.7.019.H4; CPA.7.019.vhCDR1,CPA.7.019.vhCDR2, CPA.7.019.vhCDR3, CPA.7.019.v1CDR1, CPA.7.019.v1CDR2,and CPA.7.019.v1CDR3;

CPA.7.021, CPA.7.021.VH, CPA.7.021.VL, CPA.7.021.HC, CPA.7.021.LC,CPA.7.021.H1, CPA.7.021.H2, CPA.7.021.H3 CPA.7.021.H4; CPA.7.021.vhCDR1,CPA.7.021.vhCDR2, CPA.7.021.vhCDR3, CPA.7.021.vCDR1, CPA.7.021.v1CDR2,and CPA.7.021.v1CDR3;

CPA.7.022, CPA.7.022.VH, CPA.7.022.VL, CPA.7.022.HC, CPA.7.022.LC,CPA.7.022.H1, CPA.7.022.H2, CPA.7.022.H3 CPA.7.022.H4; CPA.7.022.vhCDR1,CPA.7.022.vhCDR2, CPA.7.002201.vhCDR3, CPA.7.022.vCDR1,CPA.7.022.v1CDR2, and CPA.7.022.v1CDR3;

CPA.7.023, CPA.7.023.VH, CPA.7.023.VL, CPA.7.023.HC, CPA.7.023.LC,CPA.7.023.H1, CPA.7.023.H2, CPA.7.023.H3 CPA.7.023.H4; CPA.7.023.vhCDR1,CPA.7.023.vhCDR2, CPA.7.023.vhCDR3, CPA.7.023.vCDR1, CPA.7.023.v1CDR2,and CPA.7.023.v1CDR3;

CPA.7.024, CPA.7.024.VH, CPA.7.024.VL, CPA.7.024.HC, CPA.7.024.LC,CPA.7.024.H1, CPA.7.024.H2, CPA.7.024.H3 CPA.7.024.H4; CPA.7.024.vhCDR1,CPA.7.024.vhCDR2, CPA.7.024.vhCDR3, CPA.7.024.vCDR1, CPA.7.024.v1CDR2,and CPA.7.024.v1CDR3;

CPA.7.033, CPA.7.033.VH, CPA.7.033.VL, CPA.7.033.HC, CPA.7.033.LC,CPA.7.033.H1, CPA.7.033.H2, CPA.7.033.H3 CPA.7.033.H4; CPA.7.033.vhCDR1,CPA.7.033.vhCDR2, CPA.7.033.vhCDR3, CPA.7.033.v1CDR1, CPA.7.033.v1CDR2,and CPA.7.033.v1CDR3;

CPA.7.034, CPA.7.034.VH, CPA.7.034.VL, CPA.7.034.HC, CPA.7.034.LC,CPA.7.034.H1, CPA.7.034.H2, CPA.7.034.H3 CPA.7.034.H4; CPA.7.034.vhCDR1,CPA.7.034.vhCDR2, CPA.7.034.vhCDR3, CPA.7.034.v1CDR1, CPA.7.034.v1CDR2,and CPA.7.034.v1CDR3;

CPA.7.036, CPA.7.036.VH, CPA.7.036.VL, CPA.7.036.HC, CPA.7.036.LC,CPA.7.036.H1, CPA.7.036.H2, CPA.7.036.H3 CPA.7.036.H4; CPA.7.036.vhCDR1,CPA.7.036.vhCDR2, CPA.7.036.vhCDR3, CPA.7.036.vlCDR1, CPA.7.036.vlCDR2,and CPA.7.036.vlCDR3;

CPA.7.040, CPA.7.040.VH, CPA.7.040.VL, CPA.7.040.HC, CPA.7.040.LC,CPA.7.040.H1, CPA.7.040.H2, CPA.7.040.H3 and CPA.7.040.H4;CPA.7.040.vhCDR1, CPA.7.040.vhCDR2, CPA.7.040.vhCDR3, CPA.7.040.vCDR1,CPA.7.040.v1CDR2, and CPA.7.040.v1CDR3;

CPA.7.046, CPA.7.046.VH, CPA.7.046.VL, CPA.7.046.HC, CPA.7.046.LC,CPA.7.046.H1, CPA.7.046.H2, CPA.7.046.H3 CPA.7.046.H4; CPA.7.046.vhCDR1,CPA.7.046.vhCDR2, CPA.7.046.vhCDR3, CPA.7.046.vCDR1, CPA.7.046.v1CDR2,and CPA.7.046.v1CDR3;

CPA.7.047, CPA.7.047.VH, CPA.7.047.VL, CPA.7.047.HC, CPA.7.047.LC,CPA.7.047.H1, CPA.7.047.H2, CPA.7.047.H3 CPA.7.047.H4; CPA.7.047.vhCDR1,CPA.7.047.vhCDR2, CPA.7.047.vhCDR3, CPA.7.047.vCDR1,CPA.7.004701.v1CDR2, and CPA.7.047.v1CDR3;

CPA.7.049, CPA.7.049.VH, CPA.7.049.VL, CPA.7.049.HC, CPA.7.049.LC,CPA.7.049.H1, CPA.7.049.H2, CPA.7.049.H3 CPA.7.049.H4; CPA.7.049.vhCDR1,CPA.7.049.vhCDR2, CPA.7.049.vhCDR3, CPA.7.049.vCDR1, CPA.7.049.v1CDR2,and CPA.7.049.v1CDR3; and

CPA.7.050, CPA.7.050.VH, CPA.7.050.VL, CPA.7.050.HC, CPA.7.050.LC,CPA.7.050.H1, CPA.7.050.H2, CPA.7.050.H3 CPA.7.050.H4, CPA.7.050.vhCDR1,CPA.7.050.vhCDR2, CPA.7.050.vhCDR3, CPA.7.050.vlCDR1, CPA.7.050.v1CDR2,and CPA.7.050.vlCDR3.

In addition, there are a number of CPA antibodies generated herein thatbound to PVRIG but did not block the interaction of PVRIG and PVLR2 asshown in FIGS. 52A-52B, only eight of which sequences are includedherein in FIGS. 40A-40D, the components of which are:

CPA.7.028, CPA.7.028.VH, CPA.7.028.VL, CPA.7.028.HC, CPA.7.028.LC,CPA.7.028.H1, CPA.7.028.H2, CPA.7.028.H3 and CPA.7.028.H4;CPA.7.028.vhCDR1, CPA.7.028.vhCDR2, CPA.7.028.vhCDR3, CPA.7.028.v1CDR1,CPA.7.028.v1CDR2, and CPA.7.028.v1CDR3.

CPA.7.030, CPA.7.030.VH, CPA.7.030.VL, CPA.7.030.HC, CPA.7.030.LC,CPA.7.030.H1, CPA.7.030.H2, CPA.7.030.H3 and CPA.7.030.H4;CPA.7.030.vhCDR1, CPA.7.030.vhCDR2, CPA.7.030.vhCDR3, CPA.7.030.vCDR1,CPA.7.030.v1CDR2, and CPA.7.030.v1CDR3.

CPA.7.041, CPA.7.041.VH, CPA.7.041.VL, CPA.7.041.HC, CPA.7.041.LC,CPA.7.041.H1, CPA.7.041.H2, CPA.7.041.H3 and CPA.7.041.H4;CPA.7.041.vhCDR1, CPA.7.041.vhCDR2, CPA.7.041.vhCDR3, CPA.7.041.vCDR1,CPA.7.041.vCDR2, and CPA.7.041.v1CDR3.

CPA.7.016, CPA.7.016.VH, CPA.7.016.VL, CPA.7.016.HC, CPA.7.016.LC,CPA.7.016.H1, CPA.7.016.H2, CPA.7.016.H3 and CPA.7.016.H4;CPA.7.016.vhCDR1, CPA.7.016.vhCDR2, CPA.7.016.vhCDR3, CPA.7.016.v1CDR1,CPA.7.016.vCDR2, and CPA.7.016.v1CDR3.

CPA.7.020, CPA.7.020.VH, CPA.7.020.VL, CPA.7.020.HC, CPA.7.020.LC,CPA.7.020.H1, CPA.7.020.H2, CPA.7.020.H3 and CPA.7.020.H4;CPA.7.020.vhCDR1, CPA.7.020.vhCDR2, CPA.7.020.vhCDR3, CPA.7.020.v1CDR1,CPA.7.020.v1CDR2, and CPA.7.020.v1CDR3.

CPA.7.038, CPA.7.038.VH, CPA.7.038.VL, CPA.7.038.HC, CPA.7.038.LC,CPA.7.038.H1, CPA.7.038.H2, CPA.7.038.H3 and CPA.7.038.H4;CPA.7.038.vhCDR1, CPA.7.038.vhCDR2, CPA.7.038.vhCDR3, CPA.7.038.v1CDR1,CPA.7.038.v1CDR2, and CPA.7.038.v1CDR3.

CPA.7.044, CPA.7.044.VH, CPA.7.044.VL, CPA.7.044.HC, CPA.7.044.LC,CPA.7.044.H1, CPA.7.044.H2, CPA.7.044.H3 and CPA.7.044.H4;CPA.7.044.vhCDR1, CPA.7.044.vhCDR2, CPA.7.044.vhCDR3, CPA.7.044.v1CDR1,CPA.7.044.v1CDR2, and CPA.7.044.v1CDR3.

CPA.7.045, CPA.7.045.VH, CPA.7.045.VL, CPA.7.045.HC, CPA.7.045.LC,CPA.7.045.H1, CPA.7.045.H2, CPA.7.045.H3 and CPA.7.045.H4;CPA.7.045.vhCDR1, CPA.7.045.vhCDR2, CPA.7.045.vhCDR3, CPA.7.045.v1CDR1,CPA.7.045.v1CDR2, and CPA.7.045.v1CDR3.

As discussed herein, the invention further provides variants of theabove components, including variants in the CDRs, as outlined above. Inaddition, variable heavy chains can be 80%, 90%, 95%, 98% or 99%identical to the “VH” sequences herein, and/or contain from 1, 2, 3, 4,5, 6, 7, 8, 9, 10 amino acid changes, or more, when Fc variants areused. Variable light chains are provided that can be 80%, 90%, 95%, 98%or 99% identical to the “VL” sequences herein, and/or contain from 1, 2,3, 4, 5, 6, 7, 8, 9, 10 amino acid changes, or more, when Fc variantsare used. Similarly, heavy and light chains are provided that are 80%,90%, 95%, 98% or 99% identical to the “HC” and “LC” sequences herein,and/or contain from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid changes, ormore, when Fc variants are used.

Furthermore, the present invention provides a number of CHA antibodies,which are murine antibodies generated from hybridomas. As is well knownthe art, the six CDRs are useful when put into either human frameworkvariable heavy and variable light regions or when the variable heavy andlight domains are humanized.

Accordingly, the present invention provides antibodies, usually fulllength or scFv domains, that comprise the following CHA sets of CDRs,the sequences of which are shown in FIGS. 41A-41DD:

CHA.7.502.vhCDR1, CHA.7.502.vhCDR2, CHA.7.502.vhCDR3, CHA.7.502.vlCDR1,CHA.7.502.vlCDR2, and CHA.7.502.vCDR3.

CHA.7.503.vhCDR1, CHA.7.503.vhCDR2, CHA.7.503.vhCDR3, CHA.7.503.vlCDR1,CHA.7.503.vlCDR2, and CHA.7.503.vCDR3.

CHA.7.506.vhCDR1, CHA.7.506.vhCDR2, CHA.7.506.vhCDR3, CHA.7.506.vlCDR1,CHA.7.506.vlCDR2, and CHA.7.506.vCDR3.

CHA.7.508.vhCDR1, CHA.7.508.vhCDR2, CHA.7.508.vhCDR3, CHA.7.508.vlCDR1,CHA.7.508.vlCDR2, and CHA.7.508.vCDR3.

CHA.7.510.vhCDR1, CHA.7.510.vhCDR2, CHA.7.510.vhCDR3, CHA.7.510.vlCDR1,CHA.7.510.vlCDR2, and CHA.7.510.vCDR3.

CHA.7.512.vhCDR1, CHA.7.512.vhCDR2, CHA.7.512.vhCDR3, CHA.7.512.vlCDR1,CHA.7.512.vlCDR2, and CHA.7.512.vCDR3.

CHA.7.514.vhCDR1, CHA.7.514.vhCDR2, CHA.7.514.vhCDR3, CHA.7.514.vlCDR1,CHA.7.514.vlCDR2, and CHA.7.514.vCDR3.

CHA.7.516.vhCDR1, CHA.7.516.vhCDR2, CHA.7.516.vhCDR3, CHA.7.516.v1CDR1,CHA.7.516.v1CDR2, and CHA.7.516.vCDR3.

CHA.7.518.vhCDR1, CHA.7.518.vhCDR2, CHA.7.518.vhCDR3, CHA.7.518.v1CDR1,CHA.7.518.v1CDR2, and CHA.7.518.vCDR3.

CHA.7.520_1.vhCDR1, CHA.7.520_1.vhCDR2, CHA.7.520_1.vhCDR3,CHA.7.520_1.vlCDR1, CHA.7.520_1.vlCDR2, and CHA.7.520_1.vlCDR3.

CHA.7.520_2.vhCDR1, CHA.7.520_2.vhCDR2, CHA.7.520_2.vhCDR3,CHA.7.520_2.vlCDR1, CHA.7.520_2.vlCDR2, and CHA.7.520_2.vlCDR3.

CHA.7.522.vhCDR1, CHA.7.522.vhCDR2, CHA.7.522.vhCDR3, CHA.7.522.v1CDR1,CHA.7.522.v1CDR2, and CHA.7.522.vCDR3.

CHA.7.524.vhCDR1, CHA.7.524.vhCDR2, CHA.7.524.vhCDR3, CHA.7.524.v1CDR1,CHA.7.524.v1CDR2, and CHA.7.524.vCDR3.

CHA.7.526.vhCDR1, CHA.7.526.vhCDR2, CHA.7.526.vhCDR3, CHA.7.526.v1CDR1,CHA.7.526.v1CDR2, and CHA.7.526.vCDR3.

CHA.7.527.vhCDR1, CHA.7.527.vhCDR2, CHA.7.527.vhCDR3, CHA.7.527.v1CDR1,CHA.7.527.v1CDR2, and CHA.7.527.vCDR3.

CHA.7.528.vhCDR1, CHA.7.528.vhCDR2, CHA.7.528.vhCDR3, CHA.7.528.v1CDR1,CHA.7.528.v1CDR2, and CHA.7.528.vCDR3.

CHA.7.530.vhCDR1, CHA.7.530.vhCDR2, CHA.7.530.vhCDR3, CHA.7.530.v1CDR1,CHA.7.530.v1CDR2, and CHA.7.530.vCDR3.

CHA.7.534.vhCDR1, CHA.7.534.vhCDR2, CHA.7.534.vhCDR3, CHA.7.534.v1CDR1,CHA.7.534.v1CDR2, and CHA.7.534.vCDR3.

CHA.7.535.vhCDR1, CHA.7.535.vhCDR2, CHA.7.535.vhCDR3, CHA.7.535.v1CDR1,CHA.7.535.v1CDR2, and CHA.7.535.vCDR3.

CHA.7.537.vhCDR1, CHA.7.537.vhCDR2, CHA.7.537.vhCDR3, CHA.7.537.v1CDR1,CHA.7.537.v1CDR2, and CHA.7.537.vCDR3.

CHA.7.538_1.vhCDR1, CHA.7.538_1.vhCDR2, CHA.7.538_1.vhCDR3,CHA.7.538_1.vlCDR1, CHA.7.538_1.vlCDR2, and CHA.7.538_1.vlCDR3.

CHA.7.538_2.vhCDR1, CHA.7.538_2.vhCDR2, CHA.7.538_2.vhCDR3,CHA.7.538_2.vlCDR1, CHA.7.538_2.vlCDR2, and CHA.7.538_2.vlCDR3.

CHA.7.543.vhCDR1, CHA.7.543.vhCDR2, CHA.7.543.vhCDR3, CHA.7.543.vlCDR1,CHA.7.543.vlCDR2, and CHA.7.543.vlCDR3.

CHA.7.544.vhCDR1, CHA.7.544.vhCDR2, CHA.7.544.vhCDR3, CHA.7.544.vlCDR1,CHA.7.544.vlCDR2, and CHA.7.544.vlCDR3.

CHA.7.545.vhCDR1, CHA.7.545.vhCDR2, CHA.7.545.vhCDR3, CHA.7.545.vlCDR1,CHA.7.545.vlCDR2, and CHA.7.545.vlCDR3.

CHA.7.546.vhCDR1, CHA.7.546.vhCDR2, CHA.7.546.vhCDR3, CHA.7.546.vlCDR1,CHA.7.546.vlCDR2, and CHA.7.546.vlCDR3.

CHA.7.547.vhCDR1, CHA.7.547.vhCDR2, CHA.7.547.vhCDR3, CHA.7.547.vlCDR1,CHA.7.547.vlCDR2, and CHA.7.547.vlCDR3.

CHA.7.548.vhCDR1, CHA.7.548.vhCDR2, CHA.7.548.vhCDR3, CHA.7.548.vlCDR1,CHA.7.548.vlCDR2, and CHA.7.548.vlCDR3.

CHA.7.549.vhCDR1, CHA.7.549.vhCDR2, CHA.7.549.vhCDR3, CHA.7.549.vlCDR1,CHA.7.549.vlCDR2, and CHA.7.549.vlCDR3.

CHA.7.550.vhCDR1, CHA.7.550.vhCDR2, CHA.7.550.vhCDR3, CHA.7.550.vlCDR1,CHA.7.550.vlCDR2, and CHA.7.550.vlCDR3.

As above, these sets of CDRs may also be amino acid variants asdescribed above.

In addition, the framework regions of the variable heavy and variablelight chains can be humanized as is known in the art (with occasionalvariants generated in the CDRs as needed), and thus humanized variantsof the VH and VL chains of FIGS. 41A-41DD can be generated. Furthermore,the humanized variable heavy and light domains can then be fused withhuman constant regions, such as the constant regions from IgG1, IgG2,IgG3 and IgG4.

In particular, as is known in the art, murine VH and VL chains can behumanized as is known in the art, for example, using the IgBLAST programof the NCBI website, as outlined in Ye et al. Nucleic Acids Res.41:W34-W40 (2013), herein incorporated by reference in its entirety forthe humanization methods. IgBLAST takes a murine VH and/or VL sequenceand compares it to a library of known human germline sequences. As shownherein, for the humanized sequences generated herein, the databases usedwere IMGT human VH genes (F+ORF, 273 germline sequences) and IMGT humanVL kappa genes (F+ORF, 74 germline sequences). An exemplary five CHAsequences were chosen: CHA.7.518, CHA.7.530, CHA.7.538_1, CHA.7.538_2and CHA.7.524 (see FIGS. 41A-41DD for the VH and VL sequences). For thisembodiment of the humanization, human germline IGHV1-46(allele1) waschosen for all 5 as the acceptor sequence and the human heavy chainIGHJ4(allele1) joining region (J gene). For three of four (CHA.7.518,CHA.7.530, CHA.7.538_1 and CHA.7.538_2), human germlineIGKV1-39(allele 1) was chosen as the acceptor sequence and human lightchain IGKJ2(allele1) (J gene) was chosen. The J gene was chosen fromhuman joining region sequences compiled at IMGT® the internationalImMunoGeneTics information system as www.igmt.org. CDRs were definedaccording to the AbM definition (see www.bioinfo.org.uk/abs/). FIGS.88A-88I depict humanized sequences as well as some potential changes tooptimize binding to PVRIG.

Specific humanized antibodies of CHA antibodies include those shown inFIGS. 88A-881, FIGS. 89A-89E and FIG. 90. As will be appreciated bythose in the art, each humanized variable heavy (Humanized Heavy; HH)and variable light (Humanized Light, HL) sequence can be combined withthe constant regions of human IgG1, IgG2, IgG3 and IgG4. That is,CHA.7.518.HH1 is the first humanized variable heavy chain, andCHA.7.518.HH1.1 is the full length heavy chain, comprising the “HH1”humanized sequence with a IgG1 constant region (CHA.7.518.HH1.2 isCHA.7.518.HH1 with IgG2, etc,).

In some embodiments, the anti-PVRIG antibodies of the present inventioninclude anti-PVRIG antibodies wherein the VH and VL sequences ofdifferent anti-PVRIG antibodies can be “mixed and matched” to createother anti-PVRIG antibodies. PVRIG binding of such “mixed and matched”antibodies can be tested using the binding assays described above. e.g.,ELISAs). In some embodiments, when VH and VL chains are mixed andmatched, a VH sequence from a particular VH/VL pairing is replaced witha structurally similar VH sequence. Likewise, in some embodiments, a VLsequence from a particular VH/VL pairing is replaced with a structurallysimilar VL sequence. For example, the VH and VL sequences of homologousantibodies are particularly amenable for mixing and matching.

Accordingly, the antibodies of the invention comprise CDR amino acidsequences selected from the group consisting of (a) sequences as listedherein; (b) sequences that differ from those CDR amino acid sequencesspecified in (a) by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acidsubstitutions; (c) amino acid sequences having 90% or greater, 95% orgreater, 98% or greater, or 99% or greater sequence identity to thesequences specified in (a) or (b); (d) a polypeptide having an aminoacid sequence encoded by a polynucleotide having a nucleic acid sequenceencoding the amino acids as listed herein.

Additionally included in the definition of PVRIG antibodies areantibodies that share identity to the PVRIG antibodies enumeratedherein. That is, in certain embodiments, an anti-PVRIG antibodyaccording to the invention comprises heavy and light chain variableregions comprising amino acid sequences that are homologous to isolatedanti-PVRIG amino acid sequences of preferred anti-PVRIG immunemolecules, respectively, wherein the antibodies retain the desiredfunctional properties of the parent anti-PVRIG antibodies. The percentidentity between the two sequences is a function of the number ofidentical positions shared by the sequences (i.e., % homology=# ofidentical positions/total # of positions X 100), taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences. The comparison of sequencesand determination of percent identity between two sequences can beaccomplished using a mathematical algorithm, as described in thenon-limiting examples below.

The percent identity between two amino acid sequences can be determinedusing the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci.,4:11-17 (1988)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4. In addition, the percent identity betweentwo amino acid sequences can be determined using the Needleman andWunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availablecommercially), using either a Blossum 62 matrix or a PAM250 matrix, anda gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2,3, 4, 5, or 6.

Additionally or alternatively, the protein sequences of the presentinvention can further be used as a “query sequence” to perform a searchagainst public databases to, for example, identify related sequences.Such searches can be performed using the XBLAST program (version 2.0) ofAltschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST protein searchescan be performed with the XBLAST program, score=50, wordlength=3 toobtain amino acid sequences homologous to the antibody moleculesaccording to at least some embodiments of the invention. To obtaingapped alignments for comparison purposes, Gapped BLAST can be utilizedas described in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)can be used.

In general, the percentage identity for comparison between PVRIGantibodies is at least 75%, at least 80%, at least 90%, with at leastabout 95, 96, 97, 98 or 99% percent identity being preferred. Thepercentage identity may be along the whole amino acid sequence, forexample the entire heavy or light chain or along a portion of thechains. For example, included within the definition of the anti-PVRIGantibodies of the invention are those that share identity along theentire variable region (for example, where the identity is 95 or 98%identical along the variable regions), or along the entire constantregion, or along just the Fc domain.

In addition, also included are sequences that may have the identicalCDRs but changes in the variable domain (or entire heavy or lightchain). For example, PVRIG antibodies include those with CDRs identicalto those shown in FIGS. 63A-63D but whose identity along the variableregion can be lower, for example 95 or 98% percent identical.

B. PVRIG Antibodies that Compete for Binding with Enumerated Antibodies

The present invention provides not only the enumerated antibodies butadditional antibodies that compete with the enumerated antibodies (theCPA and CHA numbers enumerated herein that specifically bind to PVRIG)to specifically bind to the PVRIG molecule. As is shown in Example 11,the PVRIG antibodies of the invention “bin” into different epitope bins.There are four separate bins outlined herein; 1) the epitope bin intowhich CPA.7.002, CPA.7.003, CPA.7.005, CPA.7.007, CPA.7.010, CPA.7.012,CPA.7.015, CPA.7.016, CPA.7.017, CPA.7.019, CPA.7.020, CPA.7.021,CPA.7.024, CPA.7.028, CPA.7.032, CPA.7.033, CPA.7.036, CPA.7.037,CPA.7.038, CPA.7.043, CPA.7.046 and CPA.7.041 all fall into; 2) theepitope bin into which CPA.7.004, CPA.7.009, CPA.7.011, CPA.7.014,CPA.7.018, CPA.7.022, CPA.7.023, CPA.7.034, CPA.7.040, CPA.7.045 andCPA.7.047 all fall; 3) CPA.7.039, which defines the distinction betweenbin 1 and bin 2, in that bin 1 blocks CPA.7.039 binding and bin 2sandwiches the ligand with CPA.7.039, and bin 4) with CPA.7.050.

Thus, the invention provides anti-PVRIG antibodies that compete forbinding with antibodies that are in bin 1, with antibodies that are inbin 2, with antibodies that are inbin 3 and/or with antibodies that arein bin 4.

Additional antibodies that compete with the enumerated antibodies aregenerated, as is known in the art and generally outlined below.Competitive binding studies can be done as is known in the art,generally using SPR/Biacore® binding assays, as well as ELISA andcell-based assays.

C. Generation of Additional Antibodies

Additional antibodies to human PVRIG can be done as is well known in theart, using well known methods such as those outlined in the examples.Thus, additional anti-PVRIG antibodies can be generated by traditionalmethods such as immunizing mice (sometimes using DNA immunization, forexample, such as is used by Aldevron), followed by screening againsthuman PVRIG protein and hybridoma generation, with antibody purificationand recovery.

V. Nucleic Acid Compositions

Nucleic acid compositions encoding the anti-PVRIG antibodies of theinvention are also provided, as well as expression vectors containingthe nucleic acids and host cells transformed with the nucleic acidand/or expression vector compositions. As will be appreciated by thosein the art, the protein sequences depicted herein can be encoded by anynumber of possible nucleic acid sequences, due to the degeneracy of thegenetic code.

The nucleic acid compositions that encode the PVRIG antibodies willdepend on the format of the antibody. For traditional, tetramericantibodies containing two heavy chains and two light chains are encodedby two different nucleic acids, one encoding the heavy chain and oneencoding the light chain. These can be put into a single expressionvector or two expression vectors, as is known in the art, transformedinto host cells, where they are expressed to form the antibodies of theinvention. In some embodiments, for example when scFv constructs areused, a single nucleic acid encoding the variable heavychain-linker-variable light chain is generally used, which can beinserted into an expression vector for transformation into host cells.The nucleic acids can be put into expression vectors that contain theappropriate transcriptional and translational control sequences,including, but not limited to, signal and secretion sequences,regulatory sequences, promoters, origins of replication, selectiongenes, etc.

Preferred mammalian host cells for expressing the recombinant antibodiesaccording to at least some embodiments of the invention include ChineseHamster Ovary (CHO cells), PER.C6, HEK293 and others as is known in theart.

The nucleic acids may be present in whole cells, in a cell lysate, or ina partially purified or substantially pure form. A nucleic acid is“isolated” or “rendered substantially pure” when purified away fromother cellular components or other contaminants, e.g., other cellularnucleic acids or proteins, by standard techniques, includingalkaline/SDS treatment, CsCl banding, column chromatography, agarose gelelectrophoresis and others well known in the art.

To create a scFv gene, the V_(H)- and V_(L)-encoding DNA fragments areoperatively linked to another fragment encoding a flexible linker, e.g.,encoding the amino acid sequence (Gly4-Ser)3 (SEQ ID NO: 1), such thatthe V_(H) and V_(L) sequences can be expressed as a contiguoussingle-chain protein, with the V_(L) and V_(H) regions joined by theflexible linker (see e.g., Bird et al. (1988) Science 242:423-426;Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCaffertyet al., (1990) Nature 348:552-554).

VI. Formulations of Anti-PVRIG Antibodies

The therapeutic compositions used in the practice of the foregoingmethods can be formulated into pharmaceutical compositions comprising acarrier suitable for the desired delivery method. Suitable carriersinclude any material that when combined with the therapeutic compositionretains the anti-tumor function of the therapeutic composition and isgenerally non-reactive with the patient's immune system. Examplesinclude, but are not limited to, any of a number of standardpharmaceutical carriers such as sterile phosphate buffered salinesolutions, bacteriostatic water, and the like (see, generally,Remington's Pharmaceutical Sciences 16^(th) Edition, A. Osal., Ed.,1980). Acceptable carriers, excipients, or stabilizers are nontoxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, acetate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl orbenzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; sweeteners and other flavoring agents;fillers such as microcrystalline cellulose, lactose, com and otherstarches; binding agents; additives; coloring agents; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

In a preferred embodiment, the pharmaceutical composition that comprisesthe antibodies of the invention may be in a water-soluble form, such asbeing present as pharmaceutically acceptable salts, which is meant toinclude both acid and base addition salts. “Pharmaceutically acceptableacid addition salt” refers to those salts that retain the biologicaleffectiveness of the free bases and that are not biologically orotherwise undesirable, formed with inorganic acids such as hydrochloricacid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid andthe like, and organic acids such as acetic acid, propionic acid,glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid,succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid,cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid and the like. “Pharmaceuticallyacceptable base addition salts” include those derived from inorganicbases such as sodium, potassium, lithium, ammonium, calcium, magnesium,iron, zinc, copper, manganese, aluminum salts and the like. Particularlypreferred are the ammonium, potassium, sodium, calcium, and magnesiumsalts. Salts derived from pharmaceutically acceptable organic non-toxicbases include salts of primary, secondary, and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines and basic ion exchange resins, such as isopropylamine,trimethylamine, diethylamine, triethylamine, tripropylamine, andethanolamine. The formulations to be used for in vivo administration arepreferrably sterile. This is readily accomplished by filtration throughsterile filtration membranes or other methods.

Administration of the pharmaceutical composition comprising antibodiesof the present invention, preferably in the form of a sterile aqueoussolution, may be done in a variety of ways, including, but not limitedto subcutaneously and intravenously. Subcutaneous administration may bepreferable in some circumstances because the patient may self-administerthe pharmaceutical composition. Many protein therapeutics are notsufficiently potent to allow for formulation of a therapeuticallyeffective dose in the maximum acceptable volume for subcutaneousadministration. This problem may be addressed in part by the use ofprotein formulations comprising arginine-HCl, histidine, and polysorbate(see WO 04091658). Fc polypeptides of the present invention may be moreamenable to subcutaneous administration due to, for example, increasedpotency, improved serum half-life, or enhanced solubility.

As is known in the art, protein therapeutics are often delivered by IVinfusion or bolus. The antibodies of the present invention may also bedelivered using such methods. For example, administration may venious beby intravenous infusion with 0.9% sodium chloride as an infusionvehicle.

In addition, any of a number of delivery systems are known in the artand may be used to administer the Fc variants of the present invention.Examples include, but are not limited to, encapsulation in liposomes,microparticles, microspheres (eg. PLA/PGA microspheres), and the like.Alternatively, an implant of a porous, non-porous, or gelatinousmaterial, including membranes or fibers, may be used. Sustained releasesystems may comprise a polymeric material or matrix such as polyesters,hydrogels, poly(vinylalcohol), polylactides, copolymers of L-glutamicacid and ethyl-L-gutamate, ethylene-vinyl acetate, lactic acid-glycolicacid copolymers such as the LUPRON DEPOT®, andpoly-D-(−)-3-hydroxyburyric acid. The antibodies disclosed herein mayalso be formulated as immunoliposomes. A liposome is a small vesiclecomprising various types of lipids, phospholipids and/or surfactant thatis useful for delivery of a therapeutic agent to a mammal. Liposomescontaining the antibody are prepared by methods known in the art, suchas described in Epstein et al., 1985, Proc Natl Acad Sci USA, 82:3688;Hwang et al., 1980, Proc Natl Acad Sci USA, 77:4030; U.S. Pat. Nos.4,485,045; 4,544,545; and PCT WO 97/38731. Liposomes with enhancedcirculation time are disclosed in U.S. Pat. No. 5,013,556. Thecomponents of the liposome are commonly arranged in a bilayer formation,similar to the lipid arrangement of biological membranes. Particularlyuseful liposomes can be generated by the reverse phase evaporationmethod with a lipid composition comprising phosphatidylcholine,cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE).Liposomes are extruded through filters of defined pore size to yieldliposomes with the desired diameter. A chemotherapeutic agent or othertherapeutically active agent is optionally contained within the liposome(Gabizon et al., 1989, J National Cancer Inst 81:1484).

The antibodies may also be entrapped in microcapsules prepared bymethods including but not limited to coacervation techniques,interfacial polymerization (for example using hydroxymethylcellulose orgelatin-microcapsules, or poly-(methylmethacylate) microcapsules),colloidal drug delivery systems (for example, liposomes, albuminmicrospheres, microemulsions, nano-particles and nanocapsules), andmacroemulsions. Such techniques are disclosed in Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed., 1980.Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymer, which matrices are in the form of shaped articles,e.g. films, or microcapsules. Examples of sustained-release matricesinclude polyesters, hydrogels (for examplepoly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and gammaethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT® (whichare injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate), poly-D-(−)-3-hydroxybutyric acid, andProLease® (commercially available from Alkermes), which is amicrosphere-based delivery system composed of the desired bioactivemolecule incorporated into a matrix of poly-DL-lactide-co-gly colide(PLG).

The dosing amounts and frequencies of administration are, in a preferredembodiment, selected to be therapeutically or prophylacticallyeffective. As is known in the art, adjustments for protein degradation,systemic versus localized delivery, and rate of new protease synthesis,as well as the age, body weight, general health, sex, diet, time ofadministration, drug interaction and the severity of the condition maybe necessary, and will be ascertainable with routine experimentation bythose skilled in the art.

The concentration of the antibody in the formulation may vary from about0.1 to 100 weight %. In a preferred embodiment, the concentration of theFc variant is in the range of 0.003 to 1.0 molar. In order to treat apatient, a therapeutically effective dose of the Fc variant of thepresent invention may be administered. By “therapeutically effectivedose” herein is meant a dose that produces the effects for which it isadministered. The exact dose will depend on the purpose of thetreatment, and will be ascertainable by one skilled in the art usingknown techniques. Dosages may range from 0.0001 to 100 mg/kg of bodyweight or greater, for example 0.1, 1, 10, or 50 mg/kg of body weight,with 1 to 10 mg/kg being preferred.

VII. Methods of Using Anti-PVRIG Antibodies

Once made, the anti-PVRIG antibodies of the invention find use in anumber of different applications.

A. Therapeutic Uses

The anti-PVRIG antibodies of the invention find use in treatingpatients, such as human subjects, generally with a condition associatedwith PVRIG. The term “treatment” as used herein, refers to boththerapeutic treatment and prophylactic or preventative measures, whichin this example relates to treatment of cancer; however, also asdescribed below, uses of antibodies and pharmaceutical compositions arealso provided for treatment of infectious disease, sepsis, and/orautoimmune conditions, and/or for inhibiting an undesirable immuneactivation that follows gene therapy. Those in need of treatment includethose already with cancer as well as those in which the cancer is to beprevented. Hence, the mammal to be treated herein may have beendiagnosed as having the cancer or may be predisposed or susceptible tothe cancer. As used herein the term “treating” refers to preventing,delaying the onset of, curing, reversing, attenuating, alleviating,minimizing, suppressing, halting the deleterious effects or stabilizingof discernible symptoms of the above-described cancerous diseases,disorders or conditions. It also includes managing the cancer asdescribed above. By “manage” it is meant reducing the severity of thedisease, reducing the frequency of episodes of the disease, reducing theduration of such episodes, reducing the severity of such episodes,slowing/reducing cancer cell growth or proliferation, slowingprogression of at least one symptom, amelioration of at least onemeasurable physical parameter and the like. For example,immunostimulatory anti-PVRIG immune molecules should promote T cell orNK or cytokine immunity against target cells, e.g., cancer, infected orpathogen cells and thereby treat cancer or infectious diseases bydepleting the cells involved in the disease condition. Conversely,immunoinhibitory anti-PVRIG immune molecules should reduce T cell or NKactivity and/or or the secretion of proinflammatory cytokines which areinvolved in the disease pathology of some immune disease such asautoimmune, inflammatory or allergic conditions and thereby treat orameliorate the disease pathology and tissue destruction that may beassociated with such conditions (e.g., joint destruction associated withrheumatoid arthritis conditions).

The PVRIG antibodies of the invention are provided in therapeuticallyeffective dosages. A “therapeutically effective dosage” of an anti-PVRIGimmune molecule according to at least some embodiments of the presentinvention preferably results in a decrease in severity of diseasesymptoms, an increase in frequency and duration of disease symptom-freeperiods, an increase in lifespan, disease remission, or a prevention orreduction of impairment or disability due to the disease affliction. Forexample, for the treatment of PVRIG positive tumors, a “therapeuticallyeffective dosage” preferably inhibits cell growth or tumor growth by atleast about 20%, more preferably by at least about 40%, even morepreferably by at least about 60%, and still more preferably by at leastabout 80% relative to untreated subjects. The ability of a compound toinhibit tumor growth can be evaluated in an animal model systempredictive of efficacy in human tumors. Alternatively, this property ofa composition can be evaluated by examining the ability of the compoundto inhibit, such inhibition in vitro by assays known to the skilledpractitioner. A therapeutically effective amount of a therapeuticcompound can decrease tumor size, or otherwise ameliorate symptoms in asubject.

One of ordinary skill in the art would be able to determine atherapeutically effective amount based on such factors as the subject'ssize, the severity of the subject's symptoms, and the particularcomposition or route of administration selected.

1. Cancer Treatment

The PVRIG antibodies of the invention find particular use in thetreatment of cancer. In general, the antibodies of the invention areimmunomodulatory, in that rather than directly attack cancerous cells,the anti-PVRIG antibodies of the invention stimulate the immune system,generally by inhibiting the action of PVRIG. Thus, unlike tumor-targetedtherapies, which are aimed at inhibiting molecular pathways that arecrucial for tumor growth and development, and/or depleting tumor cells,cancer immunotherapy is aimed to stimulate the patient's own immunesystem to eliminate cancer cells, providing long-lived tumordestruction. Various approaches can be used in cancer immunotherapy,among them are therapeutic cancer vaccines to induce tumor-specific Tcell responses, and immunostimulatory antibodies (i.e. antagonists ofinhibitory receptors=immune checkpoints) to remove immunosuppressivepathways.

Clinical responses with targeted therapy or conventional anti-cancertherapies tend to be transient as cancer cells develop resistance, andtumor recurrence takes place. However, the clinical use of cancerimmunotherapy in the past few years has shown that this type of therapycan have durable clinical responses, showing dramatic impact on longterm survival. However, although responses are long term, only a smallnumber of patients respond (as opposed to conventional or targetedtherapy, where a large number of patients respond, but responses aretransient).

By the time a tumor is detected clinically, it has already evaded theimmune-defense system by acquiring immunoresistant and immunosuppressiveproperties and creating an immunosuppressive tumor microenvironmentthrough various mechanisms and a variety of immune cells.

Accordingly, the anti-PVRIG antibodies of the invention are useful intreating cancer. Due to the nature of an immuno-oncology mechanism ofaction, PVRIG does not necessarily need to be overexpressed on orcorrelated with a particular cancer type; that is, the goal is to havethe anti-PVRIG antibodies de-suppress T cell and NK cell activation,such that the immune system will go after the cancers.

“Cancer,” as used herein, refers broadly to any neoplastic disease(whether invasive or metastatic) characterized by abnormal anduncontrolled cell division causing malignant growth or tumor (e.g.,unregulated cell growth.) The term “cancer” or “cancerous” as usedherein should be understood to encompass any neoplastic disease (whetherinvasive, non-invasive or metastatic) which is characterized by abnormaland uncontrolled cell division causing malignant growth or tumor,non-limiting examples of which are described herein. This includes anyphysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer are exemplified in theworking examples and also are described within the specification.

Non-limiting examples of cancer that can be treated using anti-PVRIGantibodies include, but are not limited to, carcinoma, lymphoma,blastoma, sarcoma, and leukemia. More particular examples of suchcancers include squamous cell cancer, lung cancer (including small-celllung cancer, non-small cell lung cancer, adenocarcinoma of the lung, andsquamous carcinoma of the lung), cancer of the peritoneum,hepatocellular cancer, gastric or stomach cancer (includinggastrointestinal cancer), pancreatic cancer, glioblastoma, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breastcancer, colon cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney or renal cancer, livercancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma and various types of head and neck cancer, as well as B-celllymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL);small lymphocytic (SL) NHL; intermediate grade/follicular NHL;intermediate grade diffuse NHL; high grade immunoblastic NHL; high gradelymphoblastic NHL; high grade small non-cleaved cell NHL; bulky diseaseNHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenström'sMacroglobulinemia); chronic lymphocytic leukemia (CLL); acutelymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblasticleukemia; multiple myeloma and post-transplant lymphoproliferativedisorder (PTLD).

Other cancers amenable for treatment by the present invention include,but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, andleukemia or lymphoid malignancies. More particular examples of suchcancers include colorectal, bladder, ovarian, melanoma, squamous cellcancer, lung cancer (including small-cell lung cancer, non-small celllung cancer, adenocarcinoma of the lung, and squamous carcinoma of thelung), cancer of the peritoneum, hepatocellular cancer, gastric orstomach cancer (including gastrointestinal cancer), pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, breast cancer, colon cancer, colorectal cancer,endometrial or uterine carcinoma, salivary gland carcinoma, kidney orrenal cancer, liver cancer, prostate cancer, vulval cancer, thyroidcancer, hepatic carcinoma and various types of head and neck cancer, aswell as B-cell lymphoma (including low grade/follicular non-Hodgkin'slymphoma (NHL); small lymphocytic (SL) NHL; intermediategrade/follicular NHL; intermediate grade diffuse NHL; high gradeimmunoblastic NHL; high grade lymphoblastic NHL; high grade smallnon-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma;AIDS-related lymphoma; and Waldenström's Macroglobulinemia); chroniclymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairycell leukemia; chronic myeloblastic leukemia; and post-transplantlymphoproliferative disorder (PTLD), as well as abnormal vascularproliferation associated with phakomatoses, edema (such as thatassociated with brain tumors), and Meigs' syndrome. Preferably, thecancer is selected from the group consisting of colorectal cancer,breast cancer, rectal cancer, non-small cell lung cancer, non-Hodgkin'slymphoma (NHL), renal cell cancer, prostate cancer, liver cancer,pancreatic cancer, soft-tissue sarcoma, Kaposi's sarcoma, carcinoidcarcinoma, head and neck cancer, melanoma, ovarian cancer, mesothelioma,and multiple myeloma. In an exemplary embodiment the cancer is an earlyor advanced (including metastatic) bladder, ovarian or melanoma. Inanother embodiment the cancer is colorectal cancer. The cancerousconditions amenable for treatment of the invention include cancers thatexpress or do not express PVRIG and further include non-metastatic ornon-invasive as well as invasive or metastatic cancers wherein PVRIGexpression by immune, stromal or diseased cells suppress antitumorresponses and anti-invasive immune responses. The method of the presentinvention is particularly suitable for the treatment of vascularizedtumors.

As shown in the Examples, PVRIG is over expressed and/or correlates withtumor lymphocyte infiltration (as demonstrated by correlation to CD3,CD4, CD8 and PD-1 expression) in a number of different tumors of variousorigins, and thus is useful in treating any cancer, including but notlimited to, prostate cancer, liver cancer (HCC), colorectal cancer,ovarian cancer, endometrial cancer, breast cancer, pancreatic cancer,stomach cancer, cervical cancer, head and neck cancer, thyroid cancer,testis cancer, urothelial cancer, lung cancer, melanoma, non melanomaskin cancer (squamous and basal cell carcinoma), glioma, renal cancer(RCC), lymphoma (non-Hodgkins' lymphoma (NHL) and Hodgkin's lymphoma(HD)), Acute myeloid leukemia (AML), T cell Acute Lymphoblastic Leukemia(T-ALL), Diffuse Large B cell lymphoma, testicular germ cell tunors,mesothelioma and esophageal cancer

“Cancer therapy” herein refers to any method which prevents or treatscancer or ameliorates one or more of the symptoms of cancer. Typicallysuch therapies will comprises administration of immunostimulatoryanti-PVRIG antibodies (including antigen-binding fragments) either aloneor in combination with chemotherapy or radiotherapy or other biologicsand for enhancing the activity thereof, i.e., in individuals whereinexpression of PVRIG suppresses antitumor responses and the efficacy ofchemotherapy or radiotherapy or biologic efficacy.

2. Combination Therapies in Cancer

As is known in the art, combination therapies comprising a therapeuticantibody targeting an immunotherapy target and an additional therapeuticagent, specific for the disease condition, are showing great promise.For example, in the area of immunotherapy, there are a number ofpromising combination therapies using a chemotherapeutic agent (either asmall molecule drug or an anti-tumor antibody) with immuno-oncologyantibodies like anti-PD-1, and as such, the anti-PVRIG antibodiesoutlined herein can be substituted in the same way. Any chemotherapeuticagent exhibiting anticancer activity can be used according to thepresent invention; various non-limiting examples are described in thespecification.

The underlying scientific rationale for the dramatic increased efficacyof combination therapy claims that immune checkpoint blockade as amonotherapy will induce tumor regressions only when there ispre-existing strong anti-tumor immune response to be ‘unleashed’ whenthe pathway is blocked. However, in most patients and tumor types theendogenous anti-tumor immune responses are weak, and thus the inductionof anti-tumor immunity is required for the immune checkpoint blockade tobe effective, as shown in the FIG. 1 According to at least someembodiments of the present invention, PVRIG-specific antibodies,antibody fragments, conjugates and compositions comprising same, areused for treatment of all types of cancer in cancer immunotherapy incombination therapy.

The terms “in combination with” and “co-administration” are not limitedto the administration of said prophylactic or therapeutic agents atexactly the same time. Instead, it is meant that the anti-PVRIG antibodyand the other agent or agents are administered in a sequence and withina time interval such that they may act together to provide a benefitthat is increased versus treatment with only either anti-PVRIG antibodyof the present invention or the other agent or agents. It is preferredthat the anti-PVRIG antibody and the other agent or agents actadditively, and especially preferred that they act synergistically. Suchmolecules are suitably present in combination in amounts that areeffective for the purpose intended. The skilled medical practitioner candetermine empirically, or by considering the pharmacokinetics and modesof action of the agents, the appropriate dose or doses of eachtherapeutic agent, as well as the appropriate timings and methods ofadministration.

Accordingly, the antibodies of the present invention may be administeredconcomitantly with one or more other therapeutic regimens or agents. Theadditional therapeutic regimes or agents may be used to improve theefficacy or safety of the anti-PVRIG antibody. Also, the additionaltherapeutic regimes or agents may be used to treat the same disease or acomorbidity rather than to alter the action of the PVRIG antibody. Forexample, a PVRIG antibody of the present invention may be administeredto the patient along with chemotherapy, radiation therapy, or bothchemotherapy and radiation therapy.

The PVRIG antibodies of the present invention may be administered incombination with one or more other prophylactic or therapeutic agents,including but not limited to cytotoxic agents, chemotherapeutic agents,cytokines, growth inhibitory agents, anti-hormonal agents, kinaseinhibitors, anti-angiogenic agents, cardioprotectants, immunostimulatoryagents, immunosuppressive agents, agents that promote proliferation ofhematological cells, angiogenesis inhibitors, protein tyrosine kinase(PTK) inhibitors, or other therapeutic agents.

According to at least some embodiments, the anti PVRIG immune moleculescould be used in combination with any of the known in the art standardof care cancer treatment (as can be found, for example, inhttp://www.cancer.gov/cancertopics).

For example, the combination therapy can include an anti PVRIG antibodycombined with at least one other therapeutic or immune modulatory agent,other compounds or immunotherapies, or immunostimulatory strategy asdescribed herein. including, but not limited to, tumor vaccines,adoptive T cell therapy, Treg depletion, antibodies (e.g. bevacizumab,Erbitux), peptides, pepti-bodies, small molecules, chemotherapeuticagents such as cytotoxic and cytostatic agents (e.g. paclitaxel,cisplatin, vinorelbine, docetaxel, gemcitabine, temozolomide,irinotecan, 5FU, carboplatin), immunological modifiers such asinterferons and interleukins, immunostimulatory antibodies, growthhormones or other cytokines, folic acid, vitamins, minerals, aromataseinhibitors, RNAi, Histone Deacetylase Inhibitors, proteasome inhibitors,doxorubicin (Adriamycin), cisplatin bleomycin sulfate, carmustine,chlorambucil, and cyclophosphamide hydroxyurea which, by themselves, areonly effective at levels which are toxic or subtoxic to a patient.Cisplatin is intravenously administered as a 100 mg/dose once every fourweeks and Adriamycin is intravenously administered as a 60-75 mg/ml doseonce every 21 days.

According to at least some embodiments of the present invention,therapeutic agents that can be used in combination with anti-PVRIGantibodies are other potentiating agents that enhance anti-tumorresponses, e.g. other anti-immune checkpoint antibodies or otherpotentiating agents that are primarily geared to increase endogenousanti-tumor responses, such as Radiotherapy, Cryotherapy,Conventional/classical chemotherapy potentiating anti-tumor immuneresponses, Targeted therapy potentiating anti-tumor immune responses,Anti-angiogenic therapy, Therapeutic agents targeting immunosuppressivecells such as Tregs and MDSCs, Immunostimulatory antibodies, Cytokinetherapy, Therapeutic cancer vaccines, Adoptive cell transfer.

In some embodiments, anti-PVRIG antibodies are used in combination withBisphosphonates, especially amino-bisphosphonates (ABP), which haveshown to have anti-cancer activity. Some of the activities associatedwith ABPs are on human γδT cells that straddle the interface of innateand adaptive immunity and have potent anti-tumour activity.

Targeted therapies can also stimulate tumor-specific immune response byinducing the immunogenic death of tumor cells or by engaging immuneeffector mechanisms (Galluzzi et al, 2012, Nature Reviews—Drugdiscovery, Volume 11, pages 215-233).

According to at least some embodiments of the invention, targetedtherapies used as agents for combination with anti PVRIG immunemolecules for treatment of cancer are as described herein.

In some embodiments, anti-PVRIG antibodies are used in combination withtherapeutic agents targeting regulatory immunosuppressive cells such asregulatory T cells (Tregs) and myeloid derived suppressor cells (MDSCs).A number of commonly used chemotherapeutics exert non-specific targetingof Tregs and reduce the number or the immunosuppressive capacity ofTregs or MDSCs (Facciabene A. et al 2012 Cancer Res; 72(9) 2162-71;Byrne W L. et al 2011, Cancer Res. 71:691520; Gabrilovich D I. andNagaraj S, Nature Reviews 2009 Volume 9, pages 162-174). In this regard,metronomic therapy with some chemotherapy drugs results inimmunostimulatory rather than immunosuppressive effects, via modulationof regulatory cells. Thus, according to at least some embodiments of thepresent invention, anti-PVRIG immune molecule for cancer immunotherapyis used in combination with drugs selected from but not limited tocyclophosphamide, gemcitabine, mitoxantrone, fludarabine, fludarabine,docetaxel, paclitaxel, thalidomide and thalidomide derivatives.

In some embodiments, anti-PVRIG antibodies are used in combination withnovel Treg-specific targeting agents including: 1) depleting or killingantibodies that directly target Tregs through recognition of Treg cellsurface receptors such as anti-CD25 mAbs daclizumab, basiliximab or 2)ligand-directed toxins such as denileukin diftitox (Ontak)—a fusionprotein of human IL-2 and diphtheria toxin, or LMB-2—a fusion between anscFv against CD25 and Pseudomonas exotoxin and 3) antibodies targetingTreg cell surface receptors such as CTLA4, PD-1, OX40 and GITR or 4)antibodies, small molecules or fusion proteins targeting other NKreceptors such as previously identified.

In some embodiments, anti-PVRIG antibodies are used in combination withany of the options described below for disrupting Treg induction and/orfunction, including TLR (toll like receptors) agonists; agents thatinterfere with the adenosinergic pathway, such as ectonucleotidaseinhibitors, or inhibitors of the A2A adenosine receptor; TGF-βinhibitors, such as fresolimumab, lerdelimumab, metelimumab,trabedersen, LY2157299, LY210976; blockade of Tregs recruitment to tumortissues including chemokine receptor inhibitors, such as theCCR4/CCL2/CCL22 pathway.

In some embodiments, anti-PVRIG antibodies are used in combination withany of the options described below for inhibiting the immunosuppressivetumor microenvironment, including inhibitors of cytokines and enzymeswhich exert immunosuppressive activities, such as IDO(indoleamine-2,3-dioxygenase) inhibitors; inhibitors ofanti-inflammatory cytokines which promote an immunosuppressivemicroenvironment, such as IL-10, IL-35, IL-4 and IL-13; Bevacizumab®which reduces Tregs and favors the differentiation of DCs.

In some embodiments, anti-PVRIG antibodies are used in combination withany of the options described below for targeting MDSCs (myeloid-derivedsuppressive cells), including promoting their differentiation intomature myeloid cells that do not have suppressive functions by VitaminD3, or Vitamin A metabolites, such as retinoic acid, all-trans retinoicacid (ATRA); inhibition of MDSCs suppressive activity by COX2inhibitors, phosphodiesterase 5 inhibitors like sildenafil, ROSinhibitors such as nitroaspirin.

In some embodiments, anti-PVRIG antibodies are used in combination withimmunostimulatory antibodies or other agents which potentiate anti-tumorimmune responses (Pardoll J. Exp Med. 2012; 209(2): 201-209).Immunostimulatory antibodies promote anti-tumor immunity by directlymodulating immune functions, i.e. blocking other inhibitory targets orenhancing immunostimulatory proteins. According to at least someembodiments of the present invention, anti-PVRIG immune molecules forcancer immunotherapy is used in combination with antagonistic antibodiestargeting additional immune checkpoints including anti-CTLA4 mAbs, suchas ipilimumab, tremelimumab; anti-PD-1 such as nivolumabBMS-936558/MDX-1106/ONO-4538, AMP224, CT-011, MK-3475, anti-PDL-1antagonists such as BMS-936559/MDX-1105, MEDI4736, RG-7446/MPDL3280A;Anti-LAG-3 such as IMP-321), anti-TIM-3, anti-BTLA, anti-B7-H4,anti-B7-H3, Anti-VISTA; Agonistic antibodies targeting immunostimulatoryproteins, including anti-CD40 mAbs such as CP-870,893, lucatumumab,dacetuzumab; anti-CD137 mAbs such as BMS-663513 urelumab, PF-05082566;anti-OX40 mAbs, such as anti-OX40; anti-GITR mAbs such as TRX518;anti-CD27 mAbs, such as CDX-1127; and anti-ICOS mAbs.

[0325] n some embodiments, anti-PVRIG antibodies are used in combinationwith cytokines. A number of cytokines are in preclinical or clinicaldevelopment as agents potentiating anti-tumor immune responses forcancer immunotherapy, including among others: IL-2, IL-7, IL-12, IL-15,IL-17, IL-18 and IL-21, IL-23, IL-27, GM-CSF, IFNα (interferon α), IFNβ,and IFNγ. However, therapeutic efficacy is often hampered by severe sideeffects and poor pharmacokinetic properties. Thus, in addition tosystemic administration of cytokines, a variety of strategies can beemployed for the delivery of therapeutic cytokines and theirlocalization to the tumor site, in order to improve theirpharmacokinetics, as well as their efficacy and/or toxicity, includingantibody-cytokine fusion molecules (immunocytokines), chemicalconjugation to polyethylene glycol (PEGylation), transgenic expressionof cytokines in autologous whole tumor cells, incorporation of cytokinegenes into DNA vaccines, recombinant viral vectors to deliver cytokinegenes, etc. In the case of immunocytokines, fusion of cytokines totumor-specific antibodies or antibody fragments allows for targeteddelivery and therefore improved efficacy and pharmacokinetics, andreduced side effects.

In some embodiments, anti-PVRIG antibodies are used in combination withcancer vaccines. Therapeutic cancer vaccines allow for improved primingof T cells and improved antigen presentation, and can be used astherapeutic agents for potentiating anti-tumor immune responses (MellmanI. et al., 2011, Nature, 480:22-29; Schlom J, 2012, J Natl Cancer Inst;104:599-613).

Several types of therapeutic cancer vaccines are in preclinical andclinical development. These include for example:

1) Whole tumor cell vaccines, in which cancer cells removed duringsurgery are treated to enhance their immunogenicity, and injected intothe patient to induce immune responses against antigens in the tumorcells. The tumor cell vaccine can be autologous, i.e. a patient's owntumor, or allogeneic which typically contain two or three establishedand characterized human tumor cell lines of a given tumor type, such asthe GVAX vaccine platforms.

2) Tumor antigen vaccines, in which a tumor antigen (or a combination ofa few tumor antigens), usually proteins or peptides, are administered toboost the immune system (possibly with an adjuvant and/or with immunemodulators or attractants of dendritic cells such as GM-CSF). The tumorantigens may be specific for a certain type of cancer, but they are notmade for a specific patient.

3) Vector-based tumor antigen vaccines and DNA vaccines can be used as away to provide a steady supply of antigens to stimulate an anti-tumorimmune response. Vectors encoding for tumor antigens are injected intothe patient (possibly with proinflammatory or other attractants such asGM-CSF), taken up by cells in vivo to make the specific antigens, whichwould then provoke the desired immune response. Vectors may be used todeliver more than one tumor antigen at a time, to increase the immuneresponse. In addition, recombinant virus, bacteria or yeast vectorsshould trigger their own immune responses, which may also enhance theoverall immune response.

4) Oncolytic virus vaccines, such as OncoVex/T-VEC, which involves theintratumoral injection of replication-conditional herpes simplex viruswhich preferentially infects cancer cells. The virus, which is alsoengineered to express GM-CSF, is able to replicate inside a cancer cellcausing its lysis, releasing new viruses and an array of tumor antigens,and secreting GM-CSF in the process. Thus, such oncolytic virus vaccinesenhance DCs function in the tumor microenvironment to stimulateanti-tumor immune responses.

5) Dendritic cell vaccines (Palucka and Banchereau, 2102, Nat. Rev.Cancer, 12(4):265-277): Dendritic cells (DCs) phagocytose tumor cellsand present tumor antigens to tumor specific T cells. In this approach,DCs are isolated from the cancer patient and primed for presentingtumor-specific T cells. To this end several methods can be used: DCs areloaded with tumor cells or lysates; DCs are loaded with fusion proteinsor peptides of tumor antigens; coupling of tumor antigens toDC-targeting mAbs. The DCs are treated in the presence of a stimulatingfactor (such as GM-CSF), activated and matured ex vivo, and thenre-infused back into the patient in order provoke an immune response tothe cancer cells. Dendritic cells can also be primed in vivo byinjection of patients with irradiated whole tumor cells engineered tosecrete stimulating cytokines (such as GM-CSF). Similar approaches canbe carried out with monocytes. Sipuleucel-T (Provenge), a therapeuticcancer vaccine which has been approved for treatment of advancedprostate cancer, is an example of a dendritic cell vaccine.

In some embodiments, anti-PVRIG antibodies are used in combination withadoptive T cell therapy or adoptive cell transfer (ACT), which involvesthe ex vivo identification and expansion of autologous naturallyoccurring tumor specific T cells, which are then adoptively transferredback into the cancer patient (Restifo et al, 2013, Cancer Immunol.Immunother.62(4):727-36 (2013) Epub Dec. 4 2012). Cells that are infusedback into a patient after ex vivo expansion can traffic to the tumor andmediate its destruction. Prior to this adoptive transfer, hosts can beimmunodepleted by irradiation and/or chemotherapy. The combination oflymphodepletion, adoptive cell transfer, and a T cell growth factor(such as IL-2), can lead to prolonged tumor eradication in tumorpatients. A more novel approach involves the ex vivo geneticmodification of normal peripheral blood T cells to confer specificityfor tumor-associated antigens. For example, clones of TCRs of T cellswith particularly good anti-tumor responses can be inserted into viralexpression vectors and used to infect autologous T cells from thepatient to be treated. Another option is the use of chimeric antigenreceptors (CARs) which are essentially a chimeric immunoglobulin-TCRmolecule, also known as a T-body. CARs have antibody-like specificitiesand recognize MHC-nonrestricted structures on the surface of targetcells (the extracellular target-binding module), grafted onto the TCRintracellular domains capable of activating T cells (Restifo et alCancer Immunol. Immunother.62(4):727-36 (2013) Epub Dec. 4 2012; and Shiet al, Nature 493:111-115 2013.

The PVRIG antibodies and the one or more other therapeutic agents can beadministered in either order or simultaneously. The composition can belinked to the agent (as an immunocomplex) or can be administeredseparately from the agent. In the latter case (separate administration),the composition can be administered before, after or concurrently withthe agent or can be co-administered with other known therapies, e.g., ananti-cancer therapy, e.g., radiation.

Co-administration of the humanized anti-PVRIG immune molecules,according to at least some embodiments of the present invention withchemotherapeutic agents provides two anti-cancer agents which operatevia different mechanisms which yield a cytotoxic effect to human tumorcells. Such co-administration can solve problems due to development ofresistance to drugs or a change in the antigenicity of the tumor cellswhich would render them unreactive with the antibody. In otherembodiments, the subject can be additionally treated with an agent thatmodulates, e.g., enhances or inhibits, the expression or activity of Fcyor Fcy receptors by, for example, treating the subject with a cytokine.

Target-specific effector cells, e.g., effector cells linked tocompositions (e.g., human antibodies, multispecific and bispecificmolecules) according to at least some embodiments of the presentinvention can also be used as therapeutic agents. Effector cells fortargeting can be human leukocytes such as macrophages, neutrophils ormonocytes. Other cells include eosinophils, natural killer cells andother IgG- or IgA-receptor bearing cells. If desired, effector cells canbe obtained from the subject to be treated. The target-specific effectorcells can be administered as a suspension of cells in a physiologicallyacceptable solution. The number of cells administered can be in theorder of 10⁻⁸ to 10⁻⁹ but will vary depending on the therapeuticpurpose. In general, the amount will be sufficient to obtainlocalization at the target cell, e.g., a tumor cell expressing PVRIGproteins, and to effect cell killing e.g., by, e.g., phagocytosis.Routes of administration can also vary.

Therapy with target-specific effector cells can be performed inconjunction with other techniques for removal of targeted cells. Forexample, anti-tumor therapy using the compositions (e.g., humanantibodies, multispecific and bispecific molecules) according to atleast some embodiments of the present invention and/or effector cellsarmed with these compositions can be used in conjunction withchemotherapy. Additionally, combination immunotherapy may be used todirect two distinct cytotoxic effector populations toward tumor cellrejection. For example, anti-PVRIG immune molecules linked to anti-Fc-γRI or anti-CD3 may be used in conjunction with IgG- or IgA-receptorspecific binding agents.

Bispecific and multispecific molecules according to at least someembodiments of the present invention can also be used to modulate FcγRor FcγR levels on effector cells, such as by capping and elimination ofreceptors on the cell surface. Mixtures of anti-Fc receptors can also beused for this purpose.

The therapeutic compositions (e.g., human antibodies, alternativescaffolds multispecific and bispecific molecules and immunoconjugates)according to at least some embodiments of the present invention whichhave complement binding sites, such as portions from IgG1, -2, or -3 orIgM which bind complement, can also be used in the presence ofcomplement. In one embodiment, ex vivo treatment of a population ofcells comprising target cells with a binding agent according to at leastsome embodiments of the present invention and appropriate effector cellscan be supplemented by the addition of complement or serum containingcomplement. Phagocytosis of target cells coated with a binding agentaccording to at least some embodiments of the present invention can beimproved by binding of complement proteins. In another embodiment targetcells coated with the compositions (e.g., human antibodies,multispecific and bispecific molecules) according to at least someembodiments of the present invention can also be lysed by complement. Inyet another embodiment, the compositions according to at least someembodiments of the present invention do not activate complement.

The therapeutic compositions (e.g., human antibodies, alternativescaffolds multispecific and bispecific molecules and immunoconjugates)according to at least some embodiments of the present invention can alsobe administered together with complement. Thus, according to at leastsome embodiments of the present invention there are compositions,comprising human antibodies, multispecific or bispecific molecules andserum or complement. These compositions are advantageous in that thecomplement is located in close proximity to the human antibodies,multispecific or bispecific molecules. Alternatively, the humanantibodies, multispecific or bispecific molecules according to at leastsome embodiments of the present invention and the complement or serumcan be administered separately.

The anti-PVRIG immune molecules, according to at least some embodimentsof the present invention, can be used as neutralizing antibodies. Aneutralizing antibody (Nabs), is an antibody that is capable of bindingand neutralizing or inhibiting a specific antigen thereby inhibiting itsbiological effect. NAbs will partially or completely abrogate thebiological action of an agent by either blocking an important surfacemolecule needed for its activity or by interfering with the binding ofthe agent to its receptor on a target cell.

According to an additional aspect of the present invention thetherapeutic agents can be used to prevent pathologic inhibition of Tcell activity, such as that directed against cancer cells.

Thus, according to an additional aspect of the present invention thereis provided a method of treating cancer as recited herein, and/or forpromoting immune stimulation by administering to a subject in needthereof an effective amount of any one of the therapeutic agents and/ora pharmaceutical composition comprising any of the therapeutic agentsand further comprising a pharmaceutically acceptable diluent or carrier.

According to at least some embodiments, immune cells, preferably Tcells, can be contacted in vivo or ex vivo with the therapeutic agentsto modulate immune responses. The T cells contacted with the therapeuticagents can be any cell which expresses the T cell receptor, includingα/β and γ/δ T cell receptors. T-cells include all cells which expressCD3, including T-cell subsets which also express CD4 and CDS. T-cellsinclude both naïve and memory cells and effector cells such as CD8+cytotoxic T lymphocytes (CTL). T-cells also include cells such as Th1,Tc1, Th2, Tc2, Th3, Th9, Th17, Th22, Treg, follicular helper cells(T_(FH)) and Tr cells. T-cells also include NKT-cells iNKT, α/β NKT andγ/δ NKT cells, and similar unique classes of the T-cell lineage.

PVRIG blocking antibodies can also be used in combination withbispecific antibodies that target Fcα or Fcγ receptor-expressingeffectors cells to tumor cells (see, e.g., U.S. Pat. Nos. 5,922,845 and5,837,243). Bispecific antibodies can be used to target two separateantigens. For example anti-Fc receptor/anti-tumor antigen (e.g.,Her-2/neu) bispecific antibodies have been used to target macrophages tosites of tumor. This targeting may more effectively activate tumorspecific responses. The T cell arm of these responses would be augmentedby the use of PVRIG blockade. Alternatively, antigen may be delivereddirectly to DCs by the use of bispecific antibodies which bind to tumorantigen and a dendritic cell specific cell surface marker.

Tumors evade host immune surveillance by a large variety of mechanisms.Many of these mechanisms may be overcome by the inactivation of proteinswhich are expressed by the tumors and which are immunosuppressive. Theseinclude among others TGF-β (Kehrl, J. et al. (1986) J. Exp. Med. 163:1037-1050), IL-10 (Howard, M. & O'Garra, A. (1992) Immunology Today 13:198-200), and Fas ligand (Hahne, M. et al. (1996) Science 274:1363-1365). Antibodies to each of these entities may be used incombination with anti-PVRIG to counteract the effects of theimmunosuppressive agent and favor tumor immune responses by the host.

Other antibodies which may be used to activate host immuneresponsiveness can be used in combination with anti-PVRIG. These includemolecules on the surface of dendritic cells which activate DC functionand antigen presentation. Anti-CD40 antibodies are able to substituteeffectively for T cell helper activity (Ridge, J. et al. (1998) Nature393: 474-478) and can be used in conjunction with PVRIG antibodies (Ito,N. et al. (2000) Immunobiology 201 (5) 527-40). Activating antibodies toT cell costimulatory molecules such as OX-40 (Weinberg, A. et al. (2000)Immunol 164: 2160-2169), 4-1 BB (Melero, I. et al. (1997) NatureMedicine 3: 682-685 (1997), and ICOS (Hutloff, A. et al. (1999) Nature397: 262-266) as well as antibodies which block the activity of negativecostimulatory molecules such as CTLA-4 (e.g., U.S. Pat. No. 5,811,097,implimumab) or BTLA (Watanabe, N. et al. (2003) Nat Immunol 4:670-9),B7-H4 (Sica, G L et al. (2003) Immunity 18:849-61) PD-1 (may alsoprovide for increased levels of T cell activation.

Bone marrow transplantation is currently being used to treat a varietyof tumors of hematopoietic origin. While graft versus host disease is aconsequence of this treatment, therapeutic benefit may be obtained fromgraft vs. tumor responses.PVRIG blockade can be used to increase theeffectiveness of the donor engrafted tumor specific T cells.

There are also several experimental treatment protocols that involve exvivo activation and expansion of antigen specific T cells and adoptivetransfer of these cells into recipients in order to antigen-specific Tcells against tumor (Greenberg, R. & Riddell, S. (1999) Science 285:546-51). These methods may also be used to activate T cell responses toinfectious agents such as CMV. Ex vivo activation in the presence ofanti-PVRIG immune molecules may be expected to increase the frequencyand activity of the adoptively transferred T cells.

Optionally, antibodies to PVRIG can be combined with an immunogenicagent, such as cancerous cells, purified tumor antigens (includingrecombinant proteins, peptides, and carbohydrate molecules), cells, andcells transfected with genes encoding immune stimulating cytokines (Heet al (2004) J. Immunol. 173:4919-28). Non-limiting examples of tumorvaccines that can be used include peptides of MUC1 for treatment ofcolon cancer, peptides of MUC-1/CEA/TRICOM for the treatment of ovarycancer, or tumor cells transfected to express the cytokine GM-CSF(discussed further below).

In humans, some tumors have been shown to be immunogenic such as RCC. Itis anticipated that by raising the threshold of T cell activation byPVRIG blockade, we may expect to activate tumor responses in the host.

PVRIG blockade is likely to be most effective when combined with avaccination protocol. Many experimental strategies for vaccinationagainst tumors have been devised (see Rosenberg, S., 2000, Developmentof Cancer Vaccines, ASCO Educational Book Spring: 60-62; Logothetis, C.,2000, ASCO Educational Book Spring: 300-302; Khayat, D. 2000, ASCOEducational Book Spring: 414-428; Foon, K. 2000, ASCO Educational BookSpring: 730-738; see also Restifo, N. and Sznol, M., Cancer Vaccines,Ch. 61, pp. 3023-3043 in DeVita, V. et al. (eds.), 1997, Cancer:Principles and Practice of Oncology. Fifth Edition). In one of thesestrategies, a vaccine is prepared using autologous or allogeneic tumorcells. These cellular vaccines have been shown to be most effective whenthe tumor cells are transduced to express GM-CSF. GM-CSF has been shownto be a potent activator of antigen presentation for tumor vaccination(Dranoff et al. (1993) Proc. Natl. Acad. Sci U.S.A. 90: 3539-43).

The study of gene expression and large scale gene expression patterns invarious tumors has led to the definition of so-called tumor specificantigens (Rosenberg, S A (1999) Immunity 10: 281-7). In many cases,these tumor specific antigens are differentiation antigens expressed inthe tumors and in the cell from which the tumor arose, for examplemelanocyte antigens gp100, MAGE antigens, and Trp-2. More importantly,many of these antigens can be shown to be the targets of tumor specificT cells found in the host. PVRIG blockade may be used in conjunctionwith a collection of recombinant proteins and/or peptides expressed in atumor in order to generate an immune response to these proteins. Theseproteins are normally viewed by the immune system as self-antigens andare therefore tolerant to them. The tumor antigen may also include theprotein telomerase, which is required for the synthesis of telomeres ofchromosomes and which is expressed in more than 85% of human cancers andin only a limited number of somatic tissues (Kim, N et al. (1994)Science 266: 2011-2013). (These somatic tissues may be protected fromimmune attack by various means). Tumor antigen may also be“neo-antigens” expressed in cancer cells because of somatic mutationsthat alter protein sequence or create fusion proteins between twounrelated sequences (i.e. bcr-abl in the Philadelphia chromosome), oridiotype from B cell tumors.

Other tumor vaccines may include the proteins from viruses implicated inhuman cancers such a Human Papilloma Viruses (HPV), Hepatitis Viruses(HBV and HCV) and Kaposi's Herpes Sarcoma Virus (KHSV). Another form oftumor specific antigen which may be used in conjunction with PVRIGblockade is purified heat shock proteins (HSP) isolated from the tumortissue itself. These heat shock proteins contain fragments of proteinsfrom the tumor cells and these HSPs are highly efficient at delivery toantigen presenting cells for eliciting tumor immunity (Suot, R &Srivastava, P (1995) Science 269:1585-1588; Tamura, Y. et al. (1997)Science 278:117-120).

Dendritic cells (DC) are potent antigen presenting cells that can beused to prime antigen-specific responses. DC's can be produced ex vivoand loaded with various protein and peptide antigens as well as tumorcell extracts (Nestle, F. et al. (1998) Nature Medicine 4: 328-332). DCsmay also be transduced by genetic means to express these tumor antigensas well. DCs have also been fused directly to tumor cells for thepurposes of immunization (Kugler, A. et al. (2000) Nature Medicine6:332-336). As a method of vaccination, DC immunization may beeffectively combined with PVRIG blockade to activate more potentanti-tumor responses.

Use of the Therapeutic Agents According to at Least Some Embodiments ofthe Invention as Adjuvant for Cancer Vaccination

Immunization against tumor-associated antigens (TAAs) is a promisingapproach for cancer therapy and prevention, but it faces severalchallenges and limitations, such as tolerance mechanisms associated withself-antigens expressed by the tumor cells. Costimulatory molecules suchas B7.1 (CD80) and B7.2 (CD86) have improved the efficacy of gene-basedand cell-based vaccines in animal models and are under investigation asadjuvant in clinical trials. This adjuvant activity can be achievedeither by enhancing the costimulatory signal or by blocking inhibitorysignal that is transmitted by negative costimulators expressed by tumorcells (Neighbors et al., 2008 J Immunother;31(7):644-55).

According to at least some embodiments of the invention, any one ofpolyclonal or monoclonal antibody and/or antigen-binding fragmentsand/or conjugates containing same, and/or alternative scaffolds,specific to any one of PVRIG proteins, can be used as adjuvant forcancer vaccination. According to at least some embodiments, theinvention provides methods for improving immunization against TAAs,comprising administering to a patient an effective amount of any one ofpolyclonal or monoclonal antibody and/or antigen-binding fragmentsand/or conjugates containing same, and/or alternative scaffolds,specific to any one of PVRIG proteins.

In some embodiments the invention provides the use of PVRIG antibodiesto perform one or more of the following in a subject in need thereof:(a) upregulating pro-inflammatory cytokines; (b) increasing T-cellproliferation and/or expansion; (c) increasing interferon-γ or TNF-αproduction by T-cells; (d) increasing IL-2 secretion; (e) stimulatingantibody responses; (f) inhibiting cancer cell growth; (g) promotingantigenic specific T cell immunity; (h) promoting CD4⁺ and/or CD8⁺ Tcell activation; (i) alleviating T-cell suppression; (j) promoting NKcell activity; (k) promoting apoptosis or lysis of cancer cells; and/or(1) cytotoxic or cytostatic effect on cancer cells.

In other embodiments the invention provides the use of animmunostimulatory antibody, antigen-binding fragment or conjugatethereof according to at least some embodiments of the invention(optionally in a pharmaceutical composition) to antagonize at least oneimmune inhibitory effect of the PVRIG.

Such an antibody, antigen-binding fragment or conjugate thereofoptionally and preferably mediates at least one of the followingeffects:

(i) increases in immune response, (ii) increases in activation of uand/or γδ T cells, (iii) increases in cytotoxic T cell activity, (iv)increases in NK and/or NKT cell activity, (v) alleviation of u and/or γδT-cell suppression, (vi) increases in pro-inflammatory cytokinesecretion, (vii) increases in IL-2 secretion; (viii) increases ininterferon-γ production, (ix) increases in Th1 response, (x) decreasesin Th2 response, (xi) decreases or eliminates cell number and/oractivity of at least one of regulatory T cells (Tregs).

3. Assessment of Treatment

Generally the anti-PVRIG antibodies of the invention are administered topatients with cancer, and efficacy is assessed, in a number of ways asdescribed herein. Thus, while standard assays of efficacy can be run,such as cancer load, size of tumor, evaluation of presence or extent ofmetastasis, etc., mmuno-oncology treatments can be assessed on the basisof immune status evaluations as well. This can be done in a number ofways, including both in vitro and in vivo assays. For example,evaluation of changes in immune status (e.g. presence of ICOS+CD4+ Tcells following ipi treatment) along with “old fashioned” measurementssuch as tumor burden, size, invasiveness, LN involvement, metastasis,etc. can be done. Thus, any or all of the following can be evaluated:the inhibitory effects of PVRIG on CD4⁺ T cell activation orproliferation, CD8⁺ T (CTL) cell activation or proliferation, CD8⁺ Tcell-mediated cytotoxic activity and/or CTL mediated cell depletion, NKcell activity and NK mediated cell depletion, the potentiating effectsof PVRIG on Treg cell differentiation and proliferation and Treg- ormyeloid derived suppressor cell (MDSC)-mediated immunosuppression orimmune tolerance, and/or the effects of PVRIG on proinflammatorycytokine production by immune cells, e.g., IL-2, IFN-γ or TNF-αproduction by T or other immune cells.

In some embodiments, assessment of treatment is done by evaluatingimmune cell proliferation, using for example, CFSE dilution method, Ki67intracellular staining of immune effector cells, and 3H-Thymidineincorporation method,

In some embodiments, assessment of treatment is done by evaluating theincrease in gene expression or increased protein levels ofactivation-associated markers, including one or more of: CD25, CD69,CD137, ICOS, PD1, GITR, OX40, and cell degranulation measured by surfaceexpression of CD107A.

In general, gene expression assays are done as is known in the art. Seefor example Goodkind et al., Computers and Chem. Eng. 29(3):589 (2005),Han et al., Bioinform. Biol. Insights 11/15/15 9(Suppl. 1):29-46, Campoet al., Nod. Pathol. 2013 January; 26 suppl. 1:S97-S110, the geneexpression measurement techniques of which are expressly incorporated byreference herein.

In general, protein expression measurements are also similarly done asis known in the art, see for example, Wang et al., Recent Advances inCapillary Electrophoresis-Based Proteomic Techniques for BiomarkerDiscovery, Methods. Mol. Biol. 2013:984:1-12; Taylor et al, BioMed Res.Volume 2014, Article ID 361590, 8 pages, Becerk et al., Mutat. Res 2011June 17:722(2): 171-182, the measurement techniques of which areexpressly incorporated herein by reference.

In some embodiments, assessment of treatment is done by assessingcytotoxic activity measured by target cell viability detection viaestimating numerous cell parameters such as enzyme activity (includingprotease activity), cell membrane permeability, cell adherence, ATPproduction, co-enzyme production, and nucleotide uptake activity.Specific examples of these assays include, but are not limited to,Trypan Blue or PI staining, ⁵¹Cr or ³⁵S release method, LDH activity,MTT and/or WST assays, Calcein-AM assay, Luminescent based assay, andothers.

In some embodiments, assessment of treatment is done by assessing T cellactivity measured by cytokine production, measure either intracellularlyin culture supernatant using cytokines including, but not limited to,IFNγ, TNFα, GM-CSF, IL2, IL6, IL4, IL5, IL10, IL13 using well knowntechniques.

Accordingly, assessment of treatment can be done using assays thatevaluate one or more of the following: (i) increases in immune response,(ii) increases in activation of up and/or γδ T cells, (iii) increases incytotoxic T cell activity, (iv) increases in NK and/or NKT cellactivity, (v) alleviation of u and/or γδ T-cell suppression, (vi)increases in pro-inflammatory cytokine secretion, (vii) increases inIL-2 secretion; (viii) increases in interferon-γ production, (ix)increases in Th1 response, (x) decreases in Th2 response, (xi) decreasesor eliminates cell number and/or activity of at least one of regulatoryT cells (Tregs.

Assays to Measure Efficacy

In some embodiments, T cell activation is assessed using a MixedLymphocyte Reaction (MLR) assay as is described in EXAMPLE 23. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in immune response as measured for an example byphosphorylation or de-phosphorylation of different factors, or bymeasuring other post translational modifications. An increase inactivity indicates immunostimulatory activity. Appropriate increases inactivity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in activation of u and/or γδ T cells as measured for anexample by cytokine secretion or by proliferation or by changes inexpression of activation markers like for an example CD137, CD107a, PD1,etc. An increase in activity indicates immunostimulatory activity.Appropriate increases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in cytotoxic T cell activity as measured for an example bydirect killing of target cells like for an example cancer cells or bycytokine secretion or by proliferation or by changes in expression ofactivation markers like for an example CD137, CD107a, PD1, etc. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in NK and/or NKT cell activity as measured for an example bydirect killing of target cells like for an example cancer cells or bycytokine secretion or by changes in expression of activation markerslike for an example CD107a, etc. An increase in activity indicatesimmunostimulatory activity. Appropriate increases in activity areoutlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in up and/or γδ T-cell suppression, as measured for an exampleby cytokine secretion or by proliferation or by changes in expression ofactivation markers like for an example CD137, CD107a, PD1, etc. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in pro-inflammatory cytokine secretion as measured for exampleby ELISA or by Luminex or by Multiplex bead based methods or byintracellular staining and FACS analysis or by Alispot etc. An increasein activity indicates immunostimulatory activity. Appropriate increasesin activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in IL-2 secretion as measured for example by ELISA or byLuminex or by Multiplex bead based methods or by intracellular stainingand FACS analysis or by Alispot etc. An increase in activity indicatesimmunostimulatory activity. Appropriate increases in activity areoutlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in interferon-γ production as measured for example by ELISA orby Luminex or by Multiplex bead based methods or by intracellularstaining and FACS analysis or by Alispot etc. An increase in activityindicates immunostimulatory activity. Appropriate increases in activityare outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in Th1 response as measured for an example by cytokinesecretion or by changes in expression of activation markers. An increasein activity indicates immunostimulatory activity. Appropriate increasesin activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in Th2 response as measured for an example by cytokinesecretion or by changes in expression of activation markers. An increasein activity indicates immunostimulatory activity. Appropriate increasesin activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases cell number and/or activity of at least one of regulatory Tcells (Tregs), as measured for example by flow cytometry or by IHC. Adecrease in response indicates immunostimulatory activity. Appropriatedecreases are the same as for increases, outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in M2 macrophages cell numbers, as measured for example byflow cytometry or by IHC. A decrease in response indicatesimmunostimulatory activity. Appropriate decreases are the same as forincreases, outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in M2 macrophage pro-tumorigenic activity, as measured for anexample by cytokine secretion or by changes in expression of activationmarkers. A decrease in response indicates immunostimulatory activity.Appropriate decreases are the same as for increases, outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in N2 neutrophils increase, as measured for example by flowcytometry or by IHC. A decrease in response indicates immunostimulatoryactivity. Appropriate decreases are the same as for increases, outlinedbelow.

In one embodiment, the signaling pathway assay measures increases ordecreases in N2 neutrophils pro-tumorigenic activity, as measured for anexample by cytokine secretion or by changes in expression of activationmarkers. A decrease in response indicates immunostimulatory activity.Appropriate decreases are the same as for increases, outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in inhibition of T cell activation, as measured for an exampleby cytokine secretion or by proliferation or by changes in expression ofactivation markers like for an example CD137, CD107a, PD1, etc. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in inhibition of CTL activation as measured for an example bydirect killing of target cells like for an example cancer cells or bycytokine secretion or by proliferation or by changes in expression ofactivation markers like for an example CD137, CD107a, PD1, etc. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in up and/or γδ T cell exhaustion as measured for an exampleby changes in expression of activation markers. A decrease in responseindicates immunostimulatory activity. Appropriate decreases are the sameas for increases, outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases up and/or γδ T cell response as measured for an example bycytokine secretion or by proliferation or by changes in expression ofactivation markers like for an example CD137, CD107a, PD1, etc. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in stimulation of antigen-specific memory responses asmeasured for an example by cytokine secretion or by proliferation or bychanges in expression of activation markers like for an example CD45RA,CCR7 etc. An increase in activity indicates immunostimulatory activity.Appropriate increases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in apoptosis or lysis of cancer cells as measured for anexample by cytotoxicity assays such as for an example MTT, Cr release,Calcine AM, or by flow cytometry based assays like for an example CFSEdilution or propidium iodide staining etc. An increase in activityindicates immunostimulatory activity. Appropriate increases in activityare outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in stimulation of cytotoxic or cytostatic effect on cancercells. as measured for an example by cytotoxicity assays such as for anexample MTT, Cr release, Calcine AM, or by flow cytometry based assayslike for an example CFSE dilution or propidium iodide staining etc. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases direct killing of cancer cells as measured for an example bycytotoxicity assays such as for an example MTT, Cr release, Calcine AM,or by flow cytometry based assays like for an example CFSE dilution orpropidium iodide staining etc. An increase in activity indicatesimmunostimulatory activity. Appropriate increases in activity areoutlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases Th17 activity as measured for an example by cytokine secretionor by proliferation or by changes in expression of activation markers.An increase in activity indicates immunostimulatory activity.Appropriate increases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in induction of complement dependent cytotoxicity and/orantibody dependent cell-mediated cytotoxicity, as measured for anexample by cytotoxicity assays such as for an example MTT, Cr release,Calcine AM, or by flow cytometry based assays like for an example CFSEdilution or propidium iodide staining etc. An increase in activityindicates immunostimulatory activity. Appropriate increases in activityare outlined below.

In one embodiment, T cell activation is measured for an example bydirect killing of target cells like for an example cancer cells or bycytokine secretion or by proliferation or by changes in expression ofactivation markers like for an example CD137, CD107a, PD1, etc. ForT-cells, increases in proliferation, cell surface markers of activation(e.g. CD25, CD69, CD137, PD1), cytotoxicity (ability to kill targetcells), and cytokine production (e.g. IL-2, IL-4, IL-6, IFNγ, TNF-α,IL-10, IL-17A) would be indicative of immune modulation that would beconsistent with enhanced killing of cancer cells.

In one embodiment, NK cell activation is measured for example by directkilling of target cells like for an example cancer cells or by cytokinesecretion or by changes in expression of activation markers like for anexample CD107a, etc. For NK cells, increases in proliferation,cytotoxicity (ability to kill target cells and increases CD107a,granzyme, and perform expression), cytokine production (e.g. IFNγ andTNF), and cell surface receptor expression (e.g. CD25) would beindicative of immune modulation that would be consistent with enhancedkilling of cancer cells.

In one embodiment, γδ T cell activation is measured for example bycytokine secretion or by proliferation or by changes in expression ofactivation markers.

In one embodiment, Th1 cell activation is measured for example bycytokine secretion or by changes in expression of activation markers.

Appropriate increases in activity or response (or decreases, asappropriate as outlined above), are increases of 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95% or 98 to 99% percent over the signal ineither a reference sample or in control samples, for example testsamples that do not contain an anti-PVRIG antibody of the invention.Similarly, increases of at least one-, two-, three-, four- or five-foldas compared to reference or control samples show efficacy.

4. Treatment of Pathogen Infections

According to at least some embodiments, anti-PVRIG antibodies mayoptionally be used for treating infectious disease, for the same reasonsthat cancer can be treated: chronic infections are often characterizedby varying degrees of functional impairment of virus-specific T-cellresponses, and this defect is a principal reason for the inability ofthe host to eliminate the persisting pathogen. Although functionaleffector T cells are initially generated during the early stages ofinfection, they gradually lose function during the course of the chronicinfection as a result of persistent exposure to foreign antigen, givingrise to T cell exhaustion. Exhausted T cells express high levels ofmultiple co-inhibitory receptors such as CTLA-4, PD-1, and LAG3(Crawford et al., Curr Opin Immunol. 2009; 21:179-186; Kaufmann et al.,J. Immunol 2009; 182:5891-5897, Sharpe et al., Nat Immunol 2007;8:239-245). PD-1 overexpression by exhausted T cells was observedclinically in patients suffering from chronic viral infections includingHIV, HCV and HBV (Crawford et al., Curr Opin Immunol 2009; 21:179-186;Kaufmann et al., J. Immunol 2009; 182:5891-5897, Sharpe et al., NatImmunol 2007; 8:239-245). There has been some investigation into thispathway in additional pathogens, including other viruses, bacteria, andparasites (Hofmeyer et al., J Biomed Biotechnol. Vol 2011, Art. ID451694, Bhadra et al., Proc Nal. Acad Sci. 2011; 108(22):9196-201). Forexample, the PD-1 pathway was shown to be involved in controllingbacterial infection using a sepsis model induced by the standard cecalligation and puncture method. The absence of PD-1 in knockout miceprotected from sepsis-induced death in this model (Huang et al., PNAS2009: 106; 6303-6308).

T cell exhaustion can be reversed by blocking co-inhibitory pathwayssuch as PD-1 or CTLA-4 (Rivas et al., J. Immunol. 2009; 183:4284-91;Golden-Mason et al., J Virol. 2009; 83:9122-30; Hofmeyer et al., JBiomed Biotechnol. Vol 2011, Art. ID 451694), thus allowing restorationof anti-viral immune function. The therapeutic potential ofco-inhibition blockade for treating viral infection was extensivelystudied by blocking the PD-1/PD-L1 pathway, which was shown to beefficacious in several animal models of infection including acute andchronic simian immunodeficiency virus (SIV) infection in rhesus macaques(Valu et al., Nature 2009; 458:206-210) and in mouse models of chronicviral infection, such as lymphocytic choriomeningitis virus (LCMV)(Barber et al., Nature. 2006; 439:682-7), and Theiler's murineencephalomyelitis virus (TMEV) model in SJL/J mice (Duncan and MillerPLoS One. 2011; 6:e18548). In these models PD-1/PD-L1 blockade improvedanti-viral responses and promoted clearance of the persisting viruses.In addition, PD-1/PD-L1 blockade increased the humoral immunitymanifested as elevated production of specific anti-virus antibodies inthe plasma, which in combination with the improved cellular responsesleads to decrease in plasma viral loads and increased survival.

As used herein the term “infectious disorder and/or disease” and/or“infection”, used interchangeably, includes any disorder, disease and/orcondition caused by presence and/or growth of pathogenic biologicalagent in an individual host organism. As used herein the term“infection” comprises the disorder, disease and/or condition as above,exhibiting clinically evident illness (i.e., characteristic medicalsigns and/or symptoms of disease) and/or which is asymtomatic for muchor all of it course. As used herein the term “infection” also comprisesdisorder, disease and/or condition caused by persistence of foreignantigen that lead to exhaustion T cell phenotype characterized byimpaired functionality which is manifested as reduced proliferation andcytokine production. As used herein the term “infectious disorder and/ordisease” and/or “infection”, further includes any of the below listedinfectious disorders, diseases and/or conditions, caused by a bacterialinfection, viral infection, fungal infection and/or parasite infection.

Anti-PVRIG antibodies can be administered alone or in combination withone or more additional therapeutic agents used for treatment ofbacterial infections, viral infection, fungal infections, optionally asdescribed herein.

That is, an infected subject is administered an anti-PVRIG antibodiesthat antagonizes at least one PVRIG mediated effect on immunity, e.g.,its inhibitory effect on cytotoxic T cells or NK activity and/or itsinhibitory effect on the production of proinflammatory cytokines, orinhibits the stimulatory effect of PVRIG on TRegs thereby prompting thedepletion or killing of the infected cells or the pathogen, andpotentially resulting in disease remission based on enhanced killing ofthe pathogen or infected cells by the subject's immune cells.

5. Treatment of Sepsis

According to at least some embodiments, anti-PVRIG antibodies be usedfor treating sepsis. As used herein, the term “sepsis” or “sepsisrelated condition” encompasses Sepsis, Severe sepsis, Septic shock,Systemic inflammatory response syndrome (SIRS), Bacteremia, Septicemia,Toxemia, Septic syndrome.

Upregulation of inhibitory proteins has lately emerged as one of thecritical mechanisms underlying the immunosuppression in sepsis. ThePD-1/PDL-1 pathway, for example, appears to be a determining factor ofthe outcome of sepsis, regulating the delicate balance betweeneffectiveness and damage by the antimicrobial immune response. Duringsepsis in an experimental model, peritoneal macrophages and bloodmonocytes markedly increased PD-1 levels, which was associated with thedevelopment of cellular dysfunction (Huang et al 2009 PNAS106:6303-6308). Similarly, in patients with septic shock the expressionof PD-1 on peripheral T cells and of PDL-1 on monocytes was dramaticallyupregulated (Zhang et al 2011 Crit. Care 15:R70). Recent animal studieshave shown that blockade of the PD-1/PDL-1 pathway by anti-PD1 oranti-PDL1 antibodies improved survival in sepsis (Brahmamdam et al 2010J. Leukoc. Biol. 88:233-240; Zhang et al 2010 Critical Care 14:R220;Chang et al 2013 Critical Care 17:R85). Similarly, blockade of CTLA-4with anti-CTLA4 antibodies improved survival in sepsis (Inoue et al 2011Shock 36:38-44; Chang et al 2013 Critical Care 17:R85). Taken together,these findings suggest that blockade of inhibitory proteins, includingnegative costimulatory molecules, is a potential therapeutic approach toprevent the detrimental effects of sepsis (Goyert and Silver, J Leuk.Biol., 88(2): 225-226, 2010).

According to some embodiments, the invention provides treatment ofsepsis using anti-PVRIG antibodies, either alone or in combination withknown therapeutic agent effective for treating sepsis, such as thosetherapies that block the cytokine storm in the initial hyperinflammatoryphase of sepsis, and/or with therapies that have immunostimulatoryeffect in order to overcome the sepsis-induced immunosuppression phase.

Combination with standard of care treatments for sepsis, as recommendedby the “International Guidelines for Management of Severe Sepsis andSeptic Shock” (Dellinger et al 2013 Intensive Care Med 39:165-228), someof which are described below.

Broad spectrum antibiotics having activity against all likely pathogens(bacterial and/or fungal—treatment starts when sepsis is diagnosed, butspecific pathogen is not identified)—example Cefotaxime (Claforan®),Ticarcillin and clavulanate (Timentin®), Piperacillin and tazobactam(Zosyn®), Imipenem and cilastatin (Primaxin®), Meropenem (Merrem®),Clindamycin (Cleocin), Metronidazole (Flagyl®), Ceftriaxone (Rocephin®),Ciprofloxacin (Cipro®), Cefepime (Maxipime®), Levofloxacin (Levaquin®),Vancomycin or any combination of the listed drugs.

Vasopressors: example Norepinephrine, Dopamine, Epinephrine, vasopressin

Steroids: example: Hydrocortisone, Dexamethasone, or Fludrocortisone,intravenous or otherwise

Inotropic therapy: example Dobutamine for sepsis patients withmyocardial dysfunction

Recombinant human activated protein C (rhAPC), such as drotrecogin alfa(activated) (DrotAA).

β-blockers additionally reduce local and systemic inflammation.

Metabolic interventions such as pyruvate, succinate or high dose insulinsubstitutions.

Combination with novel potential therapies for sepsis:

Selective inhibitors of sPLA2-IIA (such as LY315920NA/S-5920).Rationale: The Group IIA secretory phospholipase A2 (sPLA2-IIA),released during inflammation, is increased in severe sepsis, and plasmalevels are inversely related to survival.

Phospholipid emulsion (such as GR270773). Rationale: Preclinical and exvivo studies show that lipoproteins bind and neutralize endotoxin, andexperimental animal studies demonstrate protection from septic deathwhen lipoproteins are administered. Endotoxin neutralization correlateswith the amount of phospholipid in the lipoprotein particles.

anti-TNF-α antibody: Rationale: Tumor necrosis factor-α (TNF-α) inducesmany of the pathophysiological signs and symptoms observed in sepsis

anti-CD14 antibody (such as IC14). Rationale: Upstream recognitionmolecules, like CD14, play key roles in the pathogenesis. Bacterial cellwall components bind to CD14 and co-receptors on myeloid cells,resulting in cellular activation and production of proinflammatorymediators. An anti-CD14 monoclonal antibody (IC14) has been shown todecrease lipopolysaccharide-induced responses in animal and human modelsof endotoxemia.

Inhibitors of Toll-like receptors (TLRs) and their downstream signalingpathways. Rationale: Infecting microbes display highly conservedmacromolecules (e.g., lipopolysaccharides, peptidoglycans) on theirsurface. When these macromolecules are recognized by pattern-recognitionreceptors (called Toll-like receptors [TLRs]) on the surface of immunecells, the host's immune response is initiated. This may contribute tothe excess systemic inflammatory response that characterizes sepsis.Inhibition of several TLRs is being evaluated as a potential therapy forsepsis, in particular TLR4, the receptor for Gram-negative bacteriaouter membrane lipopolysaccharide or endotoxin. Various drugs targetingTLR4 expression and pathway have a therapeutic potential in sepsis(Wittebole et al 2010 Mediators of Inflammation Vol 10 Article ID568396). Among these are antibodies targeting TLR4, soluble TLR4,Statins (such as Rosuvastatin®, Simvastatin®), Ketamine, nicotinicanalogues, eritoran (E5564), resatorvid (TAK242). In addition,antagonists of other TLRs such as chloroquine, inhibition of TLR-2 witha neutralizing antibody (anti-TLR-2).

Lansoprazole through its action on SOCS1 (suppressor of cytokinesecretion)

Talactoferrin or Recombinant Human Lactoferrin. Rationale: Lactoferrinis a glycoprotein with anti-infective and anti-inflammatory propertiesfound in secretions and immune cells. Talactoferrin alfa, a recombinantform of human lactoferrin, has similar properties and plays an importantrole in maintaining the gastrointestinal mucosal barrier integrity.Talactoferrin showed efficacy in animal models of sepsis, and inclinical trials in patients with severe sepsis (Guntupalli et al CritCare Med. 2013; 41(3):706-716).

Milk fat globule EGF factor VIII (MFG-E8)—a bridging molecule betweenapoptotic cells and phagocytes, which promotes phagocytosis of apoptoticcells.

Agonists of the ‘cholinergic anti-inflammatory pathway’, such asnicotine and analogues. Rationale: Stimulating the vagus nerve reducesthe production of cytokines, or immune system mediators, and blocksinflammation. This nerve “circuitry”, called the “inflammatory reflex”,is carried out through the specific action of acetylcholine, releasedfrom the nerve endings, on the α7 subunit of the nicotinic acetylcholinereceptor (7nAChR) expressed on macrophages, a mechanism termed ‘thecholinergic anti-inflammatory pathway’. Activation of this pathway viavagus nerve stimulation or pharmacologic α7 agonists prevents tissueinjury in multiple models of systemic inflammation, shock, and sepsis(Matsuda et al 2012 J Nippon Med Sch.79:4-18; Huston 2012 Surg. Infect.13:187-193).

Therapeutic agents targeting the inflammasome pathways. Rationale: Theinflammasome pathways greatly contribute to the inflammatory response insepsis, and critical elements are responsible for driving the transitionfrom localized inflammation to deleterious hyperinflammatory hostresponse (Cinel and Opal 2009 Crit. Care Med. 37:291-304; Matsuda et al2012 J Nippon Med Sch.79:4-18).

Stem cell therapy. Rationale: Mesenchymal stem cells (MSCs) exhibitmultiple beneficial properties through their capacity to home to injuredtissue, activate resident stem cells, secrete paracrine signals to limitsystemic and local inflammatory response, beneficially modulate immunecells, promote tissue healing by decreasing apoptosis in threatenedtissues and stimulating neoangiogenesis, and exhibit directantimicrobial activity. These effects are associated with reduced organdysfunction and improved survival in sepsis animal models, which haveprovided evidence that MSCs may be useful therapeutic adjuncts(Wannemuehler et al 2012 J. Surg. Res. 173:113-26).

Combination of anti-PVRIG antibody with other immunomodulatory agents,such as immunostimulatory antibodies, cytokine therapy, immunomodulatorydrugs. Such agents bring about increased immune responsiveness,especially in situations in which immune defenses (whether innate and/oradaptive) have been degraded, such as in sepsis-induced hypoinflammatoryand immunosuppressive condition. Reversal of sepsis-inducedimmunoparalysis by therapeutic agents that augments host immunity mayreduce the incidence of secondary infections and improve outcome inpatients who have documented immune suppression (Hotchkiss et al 2013Lancet Infect. Dis. 13:260-268; Payen et al 2013 Crit Care. 17:118).

Immunostimulatory antibodies promote immune responses by directlymodulating immune functions, i.e. blocking other inhibitory proteins orby enhancing costimulatory proteins. Experimental models of sepsis haveshown that immunostimulation by antibody blockade of inhibitoryproteins, such as PD-1, PDL-1 or CTLA-4 improved survival in sepsis(Brahmamdam et al 2010 J. Leukoc. Biol. 88:233-240; Zhang et al 2010Critical Care 14:R220; Chang et al 2013 Critical Care 17:R85; Inoue etal 2011 Shock 36:38-44), pointing to such immunostimulatory agents aspotential therapies for preventing the detrimental effects ofsepsis-induced immunosuppression (Goyert and Silver J Leuk. Biol.88(2):225-226, 2010). Immunostimulatory antibodies include: 1)Antagonistic antibodies targeting inhibitory immune checkpoints includeanti-CTLA4 mAbs (such as ipilimumab, tremelimumab), Anti-PD-1 (such asnivolumab BMS-936558/MDX-1106/ONO-4538, AMP224, CT-011, lambrozilumabMK-3475), Anti-PDL-1 antagonists (such as BMS-936559/MDX-1105, MEDI4736,RG-7446/MPDL3280A); Anti-LAG-3 such as IMP-321), Anti-TIM-3, Anti-BTLA,Anti-B7-H4, Anti-B7-H3, anti-VISTA. 2) Agonistic antibodies enhancingimmunostimulatory proteins include Anti-CD40 mAbs (such as CP-870,893,lucatumumab, dacetuzumab), Anti-CD137 mAbs (such as BMS-663513 urelumab,PF-05082566), Anti-OX40 mAbs (such as Anti-OX40), Anti-GITR mAbs (suchas TRX518), Anti-CD27 mAbs (such as CDX-1127), and Anti-ICOS mAbs.

Cytokines which directly stimulate immune effector cells and enhanceimmune responses can be used in combination with anti-GEN antibody forsepsis therapy: IL-2, IL-7, IL-12, IL-15, IL-17, IL-18 and IL-21, IL-23,IL-27, GM-CSF, IFNα (interferon α), IFNβ, IFNγ. Rationale:Cytokine-based therapies embody a direct attempt to stimulate thepatient's own immune system. Experimental models of sepsis have shownadministration of cytokines, such as IL-7 and IL-15, promote T cellviability and result in improved survival in sepsis (Unsinger et al 2010J. Immunol. 184:3768-3779; Inoue et al 2010 J. Immunol. 184:1401-1409).Interferon-γ (IFNγ) reverses sepsis-induced immunoparalysis of monocytesin vitro. An in vivo study showed that IFNγ partially reversesimmunoparalysis in vivo in humans. IFNγ and granulocyte-macrophagecolony-stimulating factor (GM-CSF) restore immune competence of ex vivostimulated leukocytes of patients with sepsis (Mouktaroudi et al CritCare. 2010; 14: P17; Leentjens et al Am J Respir Crit Care Med Vol 186,pp 838-845, 2012).

Immunomodulatory drugs such as thymosin al. Rationale: Thymosin α 1(Tα1) is a naturally occurring thymic peptide which acts as anendogenous regulator of both the innate and adaptive immune systems. Itis used worldwide for treating diseases associated with immunedysfunction including viral infections such as hepatitis B and C,certain cancers, and for vaccine enhancement. Notably, recentdevelopment in immunomodulatory research has indicated the beneficialeffect of Tal treatment in septic patients (Wu et al. Critical Care2013, 17:R8).

In the above-described sepsis therapies preferably a subject with sepsisor at risk of developing sepsis because of a virulent infection, e.g.,one resistant to antibiotics or other drugs, will be administered animmunostimulatory anti-PVRIG antibody or antigen-binding fragmentaccording to the invention, which antibody antagonizes at least onePVRIG mediated effect on immunity, e.g., its inhibitory effect oncytotoxic T cells or NK activity and/or its inhibitory effect on theproduction of proinflammatory cytokines, or inhibits the stimulatoryeffect of PVRIG on TRegs thereby promoting the depletion or killing ofthe infected cells or the pathogen, and potentially resulting in diseaseremission based on enhanced killing of the pathogen or infected cells bythe subject's endogenous immune cells. Because sepsis may rapidly resultin organ failure, in this embodiment it may be beneficial to administeranti-PVRIG antibody fragments such as Fabs rather than intact antibodiesas they may reach the site of sepsis and infection quicker than intactantibodies. In such treatment regimens antibody half-life may be oflesser concern due to the sometimes rapid morbidity of this disease.

B. Diagnostic Uses

The anti-PVRIG antibodies provided also find use in the in vitro or invivo diagnosis, including imaging, of tumors that over-express PVRIG. Itshould be noted, however, that as discussed herein, PVRIG, as animmuno-oncology target protein, is not necessarily overexpressed oncancer cells rather within the immune infiltrates in the cancer. In someinstances it is; rather, the mechanism of action, activation of immunecells such as T cells and NK cells, that results in cancer diagnosis.Accordingly, anti-PVRIG antibodies can be used to diagnose cancer.

In particular, immune cells infiltrating the tumors that over expressPVRIG, and thus can be diagnosed by anti-PVRIG antibodies, include, butare not limited to, prostate cancer, liver cancer (HCC), colorectalcancer, ovarian cancer, endometrial cancer, breast cancer, pancreaticcancer, stomach cancer, cervical cancer, head and neck cancer, thyroidcancer, testis cancer, urothelial cancer, lung cancer, melanoma, nonmelanoma skin cancer (squamous and basal cell carcinoma), glioma, renalcancer (RCC), lymphoma (NHL or HL), Acute myeloid leukemia (AML), T cellAcute Lymphoblastic Leukemia (T-ALL), Diffuse Large B cell lymphoma,testicular germ cell tumors, mesohelioma and esophageal cancer.

Generally, diagnosis can be done in several ways. In one embodiment, atissue from a patient, such as a biopsy sample, is contacted with aPVRIG antibody, generally labeled, such that the antibody binds to theendogenous PVRIG. The level of signal is compared to that of normalnon-cancerous tissue either from the same patient or a reference sample,to determine the presence or absence of cancer. The biopsy sample can befrom a solid tumor, a blood sample (for lymphomas and leukemias such asALL, T cell lymphoma, etc).

In general, in this embodiment, the anti-PVRIG is labeled, for examplewith a fluorophore or other optical label, that is detected using afluorometer or other optical detection system as is well known in theart. In an alternate embodiment, a secondary labeled antibody iscontacted with the sample, for example using an anti-human IgG antibodyfrom a different mammal (mouse, rat, rabbit, goat, etc.) to form asandwich assay as is known in the art. Alternatively, the anti-PVRIG mAbcould be directly labeled (i.e. biotin) and detection can be done by asecondary Ab directed to the labeling agent in the art.

Once over-expression of PVRIG is seen, treatment can proceed with theadministration of an anti-PVRIG antibody according to the invention asoutlined herein.

In other embodiments, in vivo diagnosis is done. Generally, in thisembodiment, the anti-PVRIG antibody (including antibody fragments) isinjected into the patient and imaging is done. In this embodiment, forexample, the antibody is generally labeled with an optical label or anMRI label, such as a gadolinium chelate, radioactive labeling of mAb(including fragments).

In some embodiments, the antibodies described herein are used for bothdiagnosis and treatment, or for diagnosis alone. When anti-PVRIGantibodies are used for both diagnosis and treatment, some embodimentsrely on two different anti-PVRIG antibodies to two different epitopes,such that the diagnostic antibody does not compete for binding with thetherapeutic antibody, although in some cases the same antibody can beused for both. For example, this can be done using antibodies that arein different bins, e.g. that bind to different epitopes on PVRIG, suchas outlined herein. Thus included in the invention are compositionscomprising a diagnostic antibody and a therapeutic antibody, and in someembodiments, the diagnostic antibody is labeled as described herein. Inaddition, the composition of therapeutic and diagnostic antibodies canalso be co-administered with other drugs as outlined herein.

Particularly useful antibodies for use in diagnosis include, but are notlimited to these enumerated antibodies, or antibodies that utilize theCDRs with variant sequences, or those that compete for binding with anyof CPA.7.001 to CPA.7.050, and in particular, those that both bind PVRIGand block receptor binding, including, CPA.7.001, CPA.7.003, CPA.7.004,CPA.7.006, CPA.7.008, CPA.7.009, CPA.7.010, CPA.7.011, CPA.7.012,CPA.7.013, CPA.7.014, CPA.7.015, CPA.7.017, CPA.7.018, CPA.7.019,CPA.7.021, CPA.7.022, CPA.7.023, CPA.7.024, CPA.7.033, CPA.7.034,CPA.7.036, CPA.7.040, CPA.7.046, CPA.7.047, CPA.7.049, and CPA.7.050. Inaddition, those that bind but do not block can also be used, includingCPA.7.016, CAP.7.020, CPA.7.028, CPA.7.030, CPA.7.038, CPA.7.044 andCPA.7.045.

As will be appreciated by those in the art, for ex vivo or in vitroassays, murine antibodies can be used, and thus the variable heavy andlight domains from any of the following can be used for diagnosis,including the CDR sets in other formats, from CHA.7.502, CHA.7.503,CHA.7.506, CHA.7.508, CHA.7.510, CHA.7.512, CHA.7.514, CHA.7.516,CHA.7.518, CHA.7.520.1, CHA.7.520.2, CHA.7.522, CHA.7.524, CHA.7.526,CHA.7.527, CHA.7.528, CHA.7.530, CHA.7.534, CHA.7.535, CHA.7.537,CHA.7.538.1, CHA.7.538.2, CHA.7.543, CHA.7.544, CHA.7.545, CHA.7.546,CHA.7.547, CHA.7.548, CHA.7.549 and CHA.7.550.

In many embodiments, a diagnostic antibody is labeled. By “labeled”herein is meant that the antibodies disclosed herein have one or moreelements, isotopes, or chemical compounds attached to enable thedetection in a screen or diagnostic procedure. In general, labels fallinto several classes: a) immune labels, which may be an epitopeincorporated as a fusion partner that is recognized by an antibody, b)isotopic labels, which may be radioactive or heavy isotopes, c) smallmolecule labels, which may include fluorescent and colorimetric dyes, ormolecules such as biotin that enable other labeling methods, and d)labels such as particles (including bubbles for ultrasound labeling) orparamagnetic labels that allow body imagining. Labels may beincorporated into the antibodies at any position and may be incorporatedin vitro or in vivo during protein expression, as is known in the art.

Diagnosis can be done either in vivo, by administration of a diagnosticantibody that allows whole body imaging as described below, or in vitro,on samples removed from a patient. “Sample” in this context includes anynumber of things, including, but not limited to, bodily fluids(including, but not limited to, blood, urine, serum, lymph, saliva, analand vaginal secretions, perspiration and semen), as well as tissuesamples such as result from biopsies of relevant tissues.

In some embodiments, in vivo imaging is done, including but not limitedto ultrasound, CT scans, X-rays, MRI and PET scans, as well as opticaltechniques, such as those using optical labels for tumors near thesurface of the body.

In vivo imaging of diseases associated with PVRIG may be performed byany suitable technique. For example, 99Tc-labeling or labeling withanother 3-ray emitting isotope may be used to label anti-PVRIGantibodies. Variations on this technique may include the use of magneticresonance imaging (MRI) to improve imaging over gamma camera techniques.

In one embodiment, the present invention provides an in vivo imagingmethod wherein an anti-PVRIG antibody is conjugated to adetection-promoting agent, the conjugated antibody is administered to ahost, such as by injection into the bloodstream, and the presence andlocation of the labeled antibody in the host is assayed. Through thistechnique and any other diagnostic method provided herein, the presentinvention provides a method for screening for the presence ofdisease-related cells in a human patient or a biological sample takenfrom a human patient.

For diagnostic imaging, radioisotopes may be bound to an anti-PVRIGantibody either directly, or indirectly by using an intermediaryfunctional group. Useful intermediary functional groups includechelators, such as ethylenediaminetetraacetic acid anddiethylenetriaminepentaacetic acid (see for instance U.S. Pat. No.5,057,313), in such diagnostic assays involving radioisotope-conjugatedanti-PVRIG antibodies, the dosage of conjugated anti-PVRIG antibodydelivered to the patient typically is maintained at as low a level aspossible through the choice of isotope for the best combination ofminimum half-life, minimum retention in the body, and minimum quantityof isotope, which will permit detection and accurate measurement.

In addition to radioisotopes and radio-opaque agents, diagnostic methodsmay be performed using anti-PVRIG antibodies that are conjugated to dyes(such as with the biotin-streptavidin complex), contrast agents,fluorescent compounds or molecules and enhancing agents (e.g.paramagnetic ions) for magnetic resonance imaging (MRI) (see, e.g., U.S.Pat. No. 6,331,175, which describes MRI techniques and the preparationof antibodies conjugated to a MRI enhancing agent). Suchdiagnostic/detection agents may be selected from agents for use inmagnetic resonance imaging, and fluorescent compounds.

In order to load an anti-PVRIG antibody with radioactive metals orparamagnetic ions, it may be necessary to react it with a reagent havinga long tail to which are attached a multiplicity of chelating groups forbinding the ions. Such a tail may be a polymer such as a polylysine,polysaccharide, or other derivatized or derivatizable chain havingpendant groups to which can be bound chelating groups such as, e.g.,porphyrins, polyamines, crown ethers, bisthiosemicarbazones, polyoximes,and like groups known to be useful for this purpose.

Chelates may be coupled to anti-PVRIG antibodies using standardchemistries. A chelate is normally linked to an anti-PVRIG antibody by agroup that enables formation of a bond to the molecule with minimal lossof immunoreactivity and minimal aggregation and/or internalcross-linking.

Examples of potentially useful metal-chelate combinations include2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, used withdiagnostic isotopes in the general energy range of 60 to 4,000 keV, suchas ¹²⁵I, ¹²³I, ¹²⁴I, ⁶²Cu, ⁶⁴Cu, ¹⁸F, ¹¹¹In, ⁶⁷Ga, ⁹⁹Tc, ⁹⁴Tc, ¹¹C, ¹³N,⁵O, and ⁷⁶Br, for radio-imaging.

Labels include a radionuclide, a radiological contrast agent, aparamagnetic ion, a metal, a fluorescent label, a chemiluminescentlabel, an ultrasound contrast agent and a photoactive agent. Suchdiagnostic agents are well known and any such known diagnostic agent maybe used. Non-limiting examples of diagnostic agents may include aradionuclide such as ¹¹⁰In, ¹¹¹In, ¹⁷⁷Lu, ¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu,⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁹⁰Y, ⁸⁹Zr, ⁹⁴mTc, ⁹⁴Tc, ⁹⁹mTc, ¹²⁰I, ¹²³I, ¹²⁴I, ¹²⁵I,¹³¹I, ^(154_158)Gd, ³²P, ¹¹C, ¹³N, ¹⁵O, ¹⁸⁶Re, ¹⁸⁸Re, ⁵¹Mn, ⁵²mMn, ⁵⁵Co,⁷²As, ⁷⁵Br, ⁷⁶Br, ⁸²mRb, ⁸³Sr, or other γ-, β-, or positron-emitters.

Paramagnetic ions of use may include chromium (III), manganese (II),iron (III), iron (II), cobalt (II), nickel (III), copper (III),neodymium (III), samarium (III), ytterbium (III), gadolinium (III),vanadium (II), terbium (III), dysprosium (III), holmium (III) or erbium(III), Metal contrast agents may include lanthanum (III), gold (III),lead (II) or bismuth (III).

Ultrasound contrast agents may comprise liposomes, such as gas filledliposomes. Radiopaque diagnostic agents may be selected from compounds,barium compounds, gallium compounds, and thallium compounds.

These and similar chelates, when complexed with non-radioactive metals,such as manganese, iron, and gadolinium may be useful for MRI diagnosticmethods in connection with anti-PVRIG antibodies. Macrocyclic chelatessuch as NOTA, DOTA, and TETA are of use with a variety of metals andradiometals, most particularly with radionuclides of gallium, yttrium,and copper, respectively. Such metal-chelate complexes may be made verystable by tailoring the ring size to the metal of interest. Otherring-type chelates such as macrocyclic polyethers, which are of interestfor stably binding nuclides, such as 223Ra may also be suitable indiagnostic methods.

Thus, the present invention provides diagnostic anti-PVRIG antibodyconjugates, wherein the anti-PVRIG antibody conjugate is conjugated to acontrast agent (such as for magnetic resonance imaging, computedtomography, or ultrasound contrast-enhancing agent) or a radionuclidethat may be, for example, a γ-, β-, α-, Auger electron-, orpositron-emitting isotope.

Anti-PVRIG antibodies may also be useful in, for example, detectingexpression of an antigen of interest in specific cells, tissues, orserum. For diagnostic applications, the antibody typically will belabeled with a detectable moiety for in vitro assays. As will beappreciated by those in the art, there are a wide variety of suitablelabels for use in in vitro testing. Suitable dyes for use in this aspectof the invention include, but are not limited to, fluorescent lanthanidecomplexes, including those of Europium and Terbium, fluorescein,rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin,methyl-coumarins, quantum dots (also referred to as “nanocrystals”; seeU.S. Ser. No. 09/315,584, hereby incorporated by reference), pyrene,Malacite green, stilbene, Lucifer Yellow, Cascade Blue™, Texas Red, Cydyes (Cy3, Cy5, etc.), alexa dyes (including Alexa, phycoerythin,bodipy, and others described in the 6th Edition of the Molecular ProbesHandbook by Richard P. Haugland, hereby expressly incorporated byreference.

Stained tissues may then be assessed for radioactivity counting as anindicator of the amount of PVRIG-associated peptides in the tumor. Theimages obtained by the use of such techniques may be used to assessbiodistribution of PVRIG in a patient, mammal, or tissue, for example inthe context of using PVRIG as a biomarker for the presence of invasivecancer cells.

EXAMPLES

Reference is made to U.S. Ser. No. 15/048,975, filed Feb. 19, 2016,entitled “PVRIG POLYPEPTIDES AND METHODS OF TREATMENT”, claimingpriority to USSN to U.S. Ser. No. 62/118,235, filed Feb. 19, 2015, andto U.S. Ser. No. 62/141,168, filed Mar. 31, 2015, all of which areexpressly incorporated herein by reference in their entirety.

Example 1: Expression Analysis of PVRIG Proteins Example 1A

The GDS3113 data set(http://www.ncbi.nlm.nih.gov/sites/GDSbrowser?acc=GDS3113) was analyzedto identify genes with a lymphoid organ specific pattern. PVRIG wasidentified as lymphocyte specific due to high expression in primary andsecondary lymphoid organs, which include peripheral blood, bone marrow,spleen, lymph nodes, tonsil and thymus (FIG. 2). Other tissue types werenegative or showed expression at background levels. In order toinvestigate which specific cell types within the total population ofimmune cells express PVRIG, additional data sets form the GeneExpression Omnibus (www.ncbi.nlm.nih.gov/GEO) were analyzed, asdescribed in “methodology” section herein. The analysis was performed onimmune cell populations derived from peripheral blood and bone marrow.PVRIG was expressed in lymphocytes both in the B-cell linage and theT-cell linage including CD8 T-cells naïve, effector and memory (FIG. 3).In addition, PVRIG was expressed in NK cells and had the highestexpression in the iNKT population (FIG. 4). The iNKT population oflymphocytes act as potent activators of antitumor immunity whenstimulated with a synthetic agonist in experimental models. However, insome settings, iNKT cells can act as suppressors and regulators ofantitumor immunity (Clin Dev Immunol. 2012; 2012:720803). Furthermore,in early clinical trials of iNKT cell-based immunotherapy demonstratedthat the infusion of ligand-pulsed antigen presenting cells treatment ofand/or in vitro activated iNKT cells were safe and well tolerated inlung cancer and head and neck cancer (Clin Immunol. 2011 August;140(2):167-76).

A key question in regards to PVRIG expression was whether TumorInfiltrating Lymphocytes (TILs) retain expression of PVRIG in the tumormicroenvironment. Analyzing expression data of TILs form follicularlymphoma, breast cancer and colon cancer showed clear expression ofPVRIG in the TILs infiltrating the tumor. In the colon cancer examplethe specificity to the immune infiltrating cells was seen as theexpression is found only in the CD45 positive population (leukocytespecific marker), and no expression is found in EPCAM positivepopulation (epithelial specific marker) or in the CD45 negative EPCAMnegative (stromal cell population). Although the CD45 is not alymphocyte specific marker, the other expression description infers thatit is expressed on the lymphocyte population (FIG. 5A colon cancer, FIG.5B breast cancer and FIG. 5C follicular lymphoma).

The mRNA expression data shown herein indicates that PVRIG is expressedin lymphocytes and in tumor infiltrating lymphocytes (TILs). Theseresults together with PVRIG inhibitory activity propose an inhibitoryrole of the molecule in T-cells, suggesting that inhibitory antibodiesto PVRIG elevates PVRIG's suppressive role on the TILs and thus enablethe TILs to induce an immune response against cancer. As the proposedmechanism of action is directed to the TILs infiltrating the tumor,rather than direct effect on the tumor cells, any cancer with immuneinfiltration is candidate for treatment using PVRIG inhibitoryantibodies.

Methodology: Raw data is downloaded from the GEO site in SOFT format. Incases where the raw data was in MAS5 format, the data was taken withoutmanipulation. If the data was in Log MAS5 then the data was converted tolinear data. If the data was in RMA format CEL files (raw data) weredownloaded and re-analyzed using MAS5. If raw CEL files were notavailable the RMA format was used.

Data was then normalized by multiplicative according to the 95thpercentile for Affy data. Datasets analyzed: GSE49910, GSE47855,GSE39397, GSE36765, GSE27928.

Example 1B

A transcriptome reference was generated based on UCSC know genes models(http://hgdownload.cse.ucsc.edu/goldenPath/hg9/database/knownGene.txt.gz).All RNA sequencing reads were aligned to the transcriptome sequencesfirst. This alignment allowed for non-unique mapping because isoformsshare many exons. Each read was then assigned genomic coordinates andexon junctions based on the transcriptome matching. The remainingunmapped reads were aligned directly to the genome by considering one ormore exon junctions. Finally, read counts were normalized as describedby Bo et al. (Bioinformatics 2010, 26 (4): 493-500) and converted togene expression values as described by Trapnell et al (Nat Biotechnol.2010 May; 28(5):511-5).

As shown in FIG. 6, based on Genotype-Tissue Expression (GTEx) data(http://www.nature.com/ng/journal/v45/n6/full/ng.2653.html;http://www.gtexportal.org/home/), PVRIG is expressed mainly in bloodcells and to lesser extent in various normal tissues. The same resultswere observed in cancerous tissues from The Cancer Genome Atlas (TCGA)(http://cancergenome.nih.gov/) in which high expression are seen inblood cancers like B-cell lymphomas and AML (FIG. 7). A gene expressionsignature was generated for a variety of cancers and normal tissuesusing GTEx and TCGA data by identifying genes with a highly correlatedexpression pattern to PVRIG.

The correlation analysis was conducted per tumor type and onlycorrelations where both genes were expressed above 0 RPKM with at least50 samples in the same tumor type, were considered. These geneexpression signatures were tested for enrichment of interactingproteins, pathways and disease genes. Enrichment p-values werecalculated for each tumor type and the mean −log(p-value) was used torank the scoring gene sets. A clear signature of lymphocytes and T-cellswas observed in a variety of cancers, as shown in FIG. 8. For instance,the top scoring gene in protein interaction was IL2, meaning that genesknown to interact with IL2 are more correlated with PVRIG than expectedby chance across most cancers. Further analysis showed that PVRIGexpression in cancer tissues are higher than normal. While in FIGS.5A-5C the median expression level of PVRIG is below 1 across most normalsolid tissues, in FIG. 6 it is clearly higher than 1 in many cancers. Asan example, when compared side by side in FIG. 7, melanoma PVRIG wasexpressed higher than normal skin (FIG. 9). We further characterized thesource of over-expression in cancer. PVRIG is highly expressed in Tcells and is highly correlated to markers of T cells in cancer. In FIG.10, PVRIG correlation to CD3, CD4 and CD8 are shown as an example inthree cancer types, namely, lung adenocarcinoma, colon adenocarcinomaand melanoma. In addition, PVRIG is highly correlated to PD1, avalidated target for immunotherapy in cancer known to be expressed on Tcells (FIG. 10).

These gene expression signatures were tested for enrichment ofinteracting proteins, pathways and disease genes. A clear signature oflymphocytes and T-cells was observed in a variety of cancers, as shownin FIG. 8. We further analyzed the correlation of PVRIG to PD1 andshowed high correlation between their expression in various tumorsincluding breast lung pancreas and kidney (Table 2). Both PD-1 and PVRIGare highly expressed on activated T cells. PVRIG showed high correlationwith T cell markers in cancer, namely, CD8A, CD4 and CD3G (FIG. 13).Taken together, these data demonstrate that cancer expression of PVRIGis associated with tumor infiltrating lymphocytes.

Methods: Genes correlation: FPKM values were transformed to log 2(FPKM+0.1). Samples with value that fulfills log 2 (FPKM+0.1)<log 2(0.1)for at least one of the genes, were omitted. Pearson CorrelationCoefficient (PCC) and the Least Squared Estimators for the regressionline were computed for the 2 lists (one list per gene). PCCs with lowervalue than 0.5 were omitted as well as PCCs that failed to showsignificant value when testing the linear correlation between theexpression levels of the 2 genes.

Gene Enrichment analysis: Pathway, interaction and disease data wereobtained from GeneGo Metacore (https://portal.genego.com), Reactome(http://www.reactome.org) and KEGG Pathways (http://www.genome.jp/kegg).To identify pathways and processes that were enriched within a givengene list, a hyper-geometric-based enrichment analysis was implemented.The hyper-geometric p-value was calculated using the R program(http://www.R-project.org) with the following command: phyper(x-l, m,n-m, k and lower.tail=FALSE), where x is the number of genes from thegene list that are members of the pathway, m is the number of genes inthe pathway, n is the total number of unique genes in all pathways, andk is the number of genes from the list that were present in at least onepathway. The resulting p-value is indicative of the likelihood ofenriching for a specific pathway by chance given the size of the genelist. The same analytical procedure was applied to gene interactionswhere all genes interacting with a given gene were treated as a pathway;or genes associated with a disease where all associated genes weretreated as a pathway. See FIGS. 64A64B.

PVRIG expression was associated with exhausted T cells in cancer. Cancersamples from TCGA were chosen that have high (4th quartile) expressionof the following 4 markers: CD8, PD-1, TIM-3 and TIGIT. Cancer sampleswere then divided to high, no change and low levels of the combinedexpression of the 4 markers. PVRIG was not detected in any of the lowexpressing markers (low or no exhausted T cells). The vast majority oftumors associated with high levels of exhausted T cells expressed highlevels of PVRIG (FIG. 22).

Example 1C

The expression of human and non-human primate PVRIG RNA and protein incell lines and primary leukocytes was evaluated.

Protocols

FACS analysis of engineered over-expressing cells: The following celllines were used to assess the specificity of anti-human PVRIGantibodies: HEK parental and HEK hPVRIG over-expressing cells. Thesecells were cultured in DMEM (Gibco)+10% fetal calf serum (Gibco)+glutamax (Gibco). For the HEK hPVRIG over-expressing cells, 0.5 μg/mlpuromycin (Gibco) was also added to the media for positive selection.For FACS analysis, all cell lines were harvested in log phase growth and50,000-100,000 cells per well were seeded in 96 well plates. Anti-humanPVRIG antibodies (human IgG1, hIgG1) and their respective controls wereadded in single point dilutions (5 μg/ml), or as an 8 point titrationseries starting at 30 μg/ml on ice for 30 mins-1 hr. The titrationseries were conducted as either 1:3 or 1:3.3 fold serial dilutions. Datawas acquired using a FACS Canto II (BD Biosciences) and analyzed usingFlowJo (Treestar) and Prism (Graphpad) software.

FACS analysis of human cell lines: The following cell lines were used toassess the expression and specificity of anti-human PVRIG antibodies:Jurkat, CA46, NK-92, OV-90, HepG2, and NCI-H441. Jurkat, CA46, andNCI-H441 cells were cultured in RPMI media+10% fetal calf serum,glutamax, non-essential amino acids (Gibco), sodium pyruvate (Gibco),and penicillin/streptomycin (Gibco). NK-92 cells were cultured in RPMImedia+25% fetal calf serum, glutamax, non-essential amino acids, sodiumpyruvate, penicillin/streptomycin, and 500 U/ml IL-2 (R&D systems).OV-90 cells were cultured in a 1:1 mixture of MCDB 105 media (Sigma)containing a final concentration of 1.5 g/L sodium bicarbonate (LifeTechnologies) and Media 199 (Sigma) containing a final concentration of2.2 g/L sodium bicarbonate with a final concentration of 15% fetal calfserum. HepG2 cells were cultured in DMEM+10% fetal calf serum+ glutamax.For FACS analysis, all cell lines were harvested in log phase growth and50,000-100,000 cells per well were seeded in 96 well plates. Anti-humanPVRIG antibodies (hIgG1) and their respective controls were added insingle point dilutions (5 μg/ml), or as an 8 point titration seriesstarting at 30 μg/ml on ice for 30 mins-1 hr. The titration series wereconducted as either 1:3 or 1:3.3 fold serial dilutions. Data wasacquired using a FACS Canto II and analyzed using FlowJo and Prismsoftware.

FACS analysis of naïve human primary leukocytes: Primary leukocytes wereobtained by Ficoll (GE Healthcare) gradient isolation of peripheralblood (Stanford Blood Bank). Leukocytes as isolated peripheral bloodmononuclear cells (PBMC) were frozen down in liquid nitrogen at adensity between 1×107 and 5×107 cells/ml in a 10% DMSO (Sigma), 90%fetal calf serum mixture. To assess protein expression of PVRIG on PBMC,antibody cocktails towards major immune subsets were designed thatincluded human anti-PVRIG antibodies. Anti-human PVRIG antibodies(hIgG1) and their respective controls were added in single pointdilutions (5 μg/ml), or in some cases, as an 8 point titration seriesstarting at 10 or 30 μg/ml on ice for 30 mins-1 hr.

Briefly, antibody cocktail mixtures were added to resuscitated PBMC thatwere seeded at 5×10⁵-1×10⁶ cells/well upon prior Fc receptor blockadeand live/dead staining (Aqua Live/Dead, Life Technologies). Antibodycocktails were incubated with PBMC for 30 mins-1 hr on ice. PBMC werethen washed and data was acquired by FACS using a FACS Canto II. Datawas analysed using FlowJo and Prism software. Immune subsets that wereanalysed include CD56 dim NK cells, CD56 bright NK cells, CD4+ T cells,CD8+ T cells, non-conventional T cells (e.g. NKT cells and γδ T cells),B cells, and monocytes.

FACS analysis of activated human effector lymphocytes: In some cases,expression of PVRIG was assessed on activated effector lymphocytesubsets either isolated from whole PBMC or in whole PBMC preparations.Effector lymphocytes were stimulated with combinations of cytokines,combinations of antibodies and cytokines, or pathogenic products. FACSanalysis of PVRIG expression on activated cells was performed analogousto that described above for naïve primary leukocytes.

To study PVRIG expression on stimulated NK cells, CD56+ cells wereisolated and cultured in various cocktails of cytokines for 1-3 days inNK cell media (RPMI+10% fetal calf serum, glutamax,penicillin/streptomycin, non-essential amino acids, sodium pyruvate, andbeta-mercaptoethanol [Gibco]). NK cells were sorted either usinganti-human CD56+ microbeads (Miltenyi Biotec) or the human NK cellisolation kit (Miltenyi Biotec) according to the manufacturer'sinstructions. Cocktails of cytokines used to simulate NK cells includedIL-2, IL-12, IL-15, IL-2/IL-12, IL-2/IL-15, IL-12/IL-15 (R&D systems).

To study PVRIG expression on stimulated T cells, CD4+ or CD8+ T cellswere isolated using CD4+ or CD8+ microbeads (Miltenyi Biotec). Theisolated cells were cultured for 3 days in the presence of variousactivating conditions in T cell media (RPMI+10% fetal calf serum,glutamax, penicillin/streptomycin, non-essential amino acids, sodiumpyruvate). Conditions used to stimulate isolated T cells include humandynabead stimulation (beads coupled to CD3/CD28 antibodies, LifeTechnologies) with IL-2 or cytokine cocktails that drive T cells tocertain phenotypes (e.g. Th1, Th2, Th17, and T regulatory phenotypes).Th1 driving cytokines are recombinant IL-12 (R&D systems) and ananti-IL-4 neutralizing antibody (Biolegend). Th2 driving conditions arerecombinant IL-4 (R&D systems) and an anti-IFN-gamma neutralizingantibody (Biolegend). Th17 driving conditions are recombinant IL-6 (R&Dsystems), TGF-beta (R&D systems), IL-23 (R&D systems), and anti-IL-4 andanti-IFNγ neutralizing antibodies. T regulatory driving conditions arerecombinant TGF-beta and IL-2, and anti-IL-4 and anti-IFNγ neutralizingantibodies.

Alternatively, activated T cells were also analyzed in whole stimulatedPBMC cultures with staphylococcal enterotoxin B (SEB) antigen (ListBiological Laboratories) for 3 days, or in a mixed lymphocyte reaction(MLR) where CD4+ T cells are co-cultured with allogeneic dendritic cellsfor 2 or 5 days.

FACS analysis of human polarized monocytes: PVRIG expression wasassessed on dendritic cells derived from polarized monocytes. In thisinstance, CD14+ cells were enriched using RosetteSep human monocyteenrichment according to manufacturer's instructions. After CD14+ cellenrichment, monocytes were polarized to dendritic cells upon culturewith GM-CSF (R&D systems) and IL-4 (R&D systems) for 4 days in RPMI+10%fetal calf serum, glutamax, penicillin/streptomycin, non-essential aminoacids, sodium pyruvate, and beta-mercaptoethanol.

RNA expression analysis of human cell lines and leukocytes by PCR: Celllines that were assessed for RNA expression by qPCR were Jurkat, CA46,Daudi, Raji, and expi 293 cells. Jurkat, CA46, Raji, and Daudi cellswere cultured in RPMI media+10% fetal calf serum, glutamax,non-essential amino acids, sodium pyruvate, and penicillin/streptomycin.Expi 293 cells were cultured in DMEM+10% FCS+ glutamax. OV-90, HepG2,and NCI-H441 RNA was analysed by a bioinformatics screen of the cancercell line atlas. For those cell lines that were assessed for RNAexpression by qPCR, the cells were harvested in log phase growth and1,000,000 cells were harvested, washed in PBS, and lysed in 350 ul ofRLT buffer (Qiagen). Lysed cells in RLT buffer were stored at −80° C.until use.

Primary leukocytes that were assessed for RNA expression were CD56+NKcells, CD4+ T cells, CD8+ T cells, and CD14+ monocytes. Cell populationswere isolated using human CD56+, CD4+, CD8+, and CD14+ positiveselection kits according to manufacturer's instructions (MiltenyiBiotec). After sorting, cells were lysed in 350 ul of RLT buffer andstored at −80° C. until use. In some instances, activated PBMC subsets(activation conditions outlined above) were harvested from culture andwere lysed in 350 ul of RLT buffer and stored at −80° C. until use.

Upon day of use, RNA was generated from lysed cells using the Qiagenmini kit according to the manufacturer's instructions. cDNA wasgenerated using Applied Biosystems high capacity cDNA reversetranscription kit. qPCR using cDNA was performed using Tagman primers(ThermoFisher) and Applied Biosystems Tagman fast advanced mastermix.The PVRIG primer set used was Tagman catalogue number: Hs04189293_g1.Beta-actin housekeeping primer set used was Tagman catalogue number:Hs01060665_g1. Expression of transcript was assessed by quantifying Ctvalues and relative expression was calculated by the 2(−ΔΔCt) method.Data was acquired on an Applied Biosystems Step One Plus instrument.

FACS analysis of cynomolgus PVRIG engineered over-expressing cells: Thefollowing cell lines were used to assess the cross-reactivity ofanti-human PVRIG antibodies with cynomolgus PVRIG (cPVRIG): expiparental and expi cPVRIG over-expressing cells. These cells werecultured in DMEM+10% fetal calf serum+ glutamax. expi cPVRIG transientover-expressing cells were generated by electroporating cPVRIG DNA intoparental expi cells using the Neon transfection system. For FACSanalysis, expi cPVRIG cells were used between 1-3 days posttransfection. Parental expi cells were harvested from log growth phase.50,000-100,000 cells of per well of each type were seeded in 96 wellplates. Anti-human PVRIG antibodies (hIgG1) and their respectivecontrols were added in single point dilutions (5 μg/ml), or as an 8point titration series starting at 100 μg/ml on ice for 30 mins-1 hr.The titration series were conducted as either 1:3 or 1:3.3 fold serialdilutions. Data was acquired using a FACS Canto II and analyzed usingFlowJo and Prism software.

FACS analysis of naïve primary cynomolgus monkey leukocytes: Primarycynomolgus monkey (cyno) leukocytes were obtained from fresh blood whichwas drawn no longer than 24 hours prior to expression analysis. Bloodwas sourced from Bioreclamation. To assess protein expression of PVRIGon cyno PBMC, antibody cocktails towards major immune subsets weredesigned that included human anti-PVRIG antibodies. Anti-human PVRIGantibodies (hIgG1) and their respective controls were added in singlepoint dilutions (5 μg/ml).

Briefly, antibody cocktail mixtures were added to PBMC that were seededat 5×10⁵-1×10⁶ cells/well upon prior Fc receptor blockade and live/deadstaining. Antibody cocktails were incubated with PBMC for 30 mins-1 hron ice. PBMC were then washed and data was acquired by FACS using a FACSCanto II. Data was analysed using Prism software. Immune subsets thatwere analysed include CD16+ lymphocytes, CD14+/CD56+ monocytes/myeloidcells, and CD3+ T cells.

RNA expression analysis of primary cynomolgus monkey leukocytes: Primaryleukocytes that were assessed for RNA expression were CD56+, CD16+, andCD56-/CD16-subsets. Cell populations were isolated using non-humanprimate CD56 and CD16 positive selection kits according tomanufacturer's instructions (Miltenyi Biotec). After sorting, cells werelysed in 350 ul of RLT buffer and stored at −80° C. until use.

Upon day of use, RNA was generated from lysed cells using the Qiagenmini kit according to the manufacturer's instructions. cDNA wasgenerated using Applied Biosystems high capacity cDNA reversetranscription kit. qPCR using cDNA was performed using Tagman primersand Applied Biosystems Tagman fast advanced mastermix. Two sets ofprimers to detect cyno PVRIG were designed by Compugen USA, Inc andmanufactured by Genscript. The sequence and primer codes are:

Primer set 1 Forward: (SEQ ID NO: 2) CTTGTGTTCACCACCTCTGG Reverse:(SEQ ID NO: 3) TGTTCTCATCGCAGGAGGTC Primer set 2 Forward: (SEQ ID NO: 4)TTGGCTGTGGATACCTCCTT Reverse: (SEQ ID NO: 5) ATAAGGGTCGTGGAGAGCAG

Beta-actin primers were used for housekeeping and the primer set usedwas Tagman catalogue number: Mf04354341_g1. Expression of transcriptswas assessed by quantifying Ct values and relative expression wascalculated by the 2^((−ΔΔCt)) method. Products generated with PVRIGprimers and beta-actin primers were also size analysed by traditionalRT-PCR using a 2.5% agarose gel. qPCR data was acquired using an AppliedBiosystems Step One Plus instrument.

Results

PVRIG antibodies recognize PVRIG on overexpressing cells: To screen forantibodies that were specific for PVRIG, we assessed the ability ofantibodies that were generated from a phage campaign to bind HEK celllines that were engineered to overexpress PVRIG. The majority ofantibodies from this campaign upon reformatting to human IgG1 bound tothe HEK hPVRIG cells, albeit with varying affinity. Furthermore, themajority of these antibodies also showed low background binding to HEKparental cell lines indicating high specificity towards PVRIG. FIG. 27shows one example of the specificity of PVRIG antibodies. A summary ofall binding characteristics of the antibodies towards HEK hPVRIG cellsrelative to control that were generated in this phage campaign aredisplayed in FIGS. 31A-31B.

Human PVRIG RNA is expressed in a range of cancer cell lines: Toinitially screen for cell lines that could be used to assess PVRIGprotein expression by antibodies, we examined the cancer cell line atlasfor cell lines that were high for PVRIG RNA as assessed bybioinformatics. We found four cell lines that were readily accessiblecommercially that were high expressors for PVRIG RNA that we chose tovalidate by qPCR analysis. These cell lines were Jurkat, CA46, Raji, andDaudi.

When qPCR analysis was conducted, we detected PVRIG RNA in all four celllines consistent with the bioinformatics analysis (FIG. 28). As anegative control we included expi cells that had relatively low PVRIGRNA expression.

Human PVRIG RNA is expressed in T cells and NK cells: To initiallyscreen PBMC for subsets likely to be positive for PVRIG protein asdetected by our antibodies, we sorted major PBMC subsets and examinedPVRIG RNA expression by qPCR. Levels of PVRIG RNA in CD56+NK cells, CD4+T cells, CD8+ T cells, and CD14+ monocytes were compared to those inJurkat, HEK parental, and HEK hPVRIG cell lines. As shown in FIG. 29,PVRIG RNA was detected most highly and up to 50 fold higher in CD4+ Tcells, CD8+ T cells, and CD56+NK cells when normalized to HEK GFP cells.Similar to FIG. 28, Jurkat cells also showed positive expression. Incontrast, CD14+ monocytes did not show higher PVRIG expression relativeto HEK GFP cells indicating very low PVRIG RNA expression.

In addition to analyzing naïve PBMC, select populations (effectorlymphocytes) were also activated under various stimulatory conditionsand expression of PVRIG RNA was assessed. More specifically, NK cellswere activated with various combinations of stimulatory cytokines,whereas T cells were polyclonally activated with human activatordynabeads or staphylococcus enterotoxin B (SEB) with or withoutpolarizing cytokines (see protocol section for details). As shown inFIGS. 30A and 30B, PVRIG RNA expression generally increased in both NKcells and T cells upon various stimulation conditions, the extent ofwhich depended on the individual donor. More specifically, FIG. 30ashows PVRIG RNA expression in naïve and activated CD4 T cells and NKcells. FIG. 30b shows PVRIG RNA expression in naïve and activated CD8 Tcells.

PVRIG antibodies recognize PVRIG protein on NK cells most prominently innaïve and activated primary immune subsets: Upon confirming the RNAexpression pattern of PVRIG RNA expression in naïve and activated PBMCsubsets, we used our panel of PVRIG antibodies to assess proteinexpression. We first assessed PVRIG expression in naïve PBMC subsets.The population which displayed the highest level of PVRIG was NK cells.CD4+ and CD8+ T cells showed low levels of PVRIG, while B cells andmonocytes had no detectable expression. A summary of expression on NKcells and CD8+ T cells as detected by our antibodies is shown in FIGS.32A-32B. Other minor subsets also displayed PVRIG expression andincluded non-conventional T cells such as NKT cells and γδ T cells. Theexpression pattern on PBMC subsets was very similar across all donors wesourced and analyzed.

When PVRIG protein was assessed after various stimulation conditions(including polyclonal simulation, cytokine stimulation, and MLR), therewas no robust up-regulation of PVRIG on any PBMC subsets, including NKcells and CD4+ and CD8+ T cells. Furthermore, monocytes which werepolarized in vitro to dendritic cells with GM-CSF and IL-4 did not showdetectable PVRIG expression consistent with that seen on non-polarizedmonocytes.

PVRIG is detected on cell lines by a proportion of PVRIG antibodies: Inaddition to screening PBMC for PVRIG protein expression, we wanted tounderstand whether it was also expressed on cancer cell lines. Using thepositive cell lines identified by RNA expression (FIG. 28), we chose toscreen our antibodies on Jurkat and CA46 cells as they showed the lowestabsolute Ct values relative to our housekeeping gene. We also chose arange of negative cell lines to further validate the specificity of ourantibodies which included OV-90, NCI-H441, and HepG2. A proportion ofour antibodies did detect PVRIG protein expression on Jurkat and CA46cells (FIGS. 31A-31B), but not the negative cell lines. An example ofPVRIG detection on Jurkat and CA46 is shown in FIG. 33 with arepresentative antibody, CPA.7.021. The expression on Jurkat and CA46was completely in accordance with each other and the intensity ofexpression was similar across the two cell lines.

PVRIG antibodies detect cynomolgus PVRIG transiently expressed on expicells: In order to assess the pre-clinical suitability of our anti-humanPVRIG antibodies for pharmacological studies in cynomolgus monkey, wewanted to understand whether our antibodies were able to cross-reactwith cynomolgus PVRIG (cPVRIG). A proportion of our antibodies were ableto detect cPVRIG which was transiently transfected onto expi cells (FIG.29). An example of an antibody that yielded negative staining(CPA.7.021) and one that yielded positive staining (CPA.7.024) are shownin FIGS. 34A-34D.

PVRIG RNA is detected in cynomolgus PBMC: Prior to assessment of PVRIGprotein on cyno PBMC, we firstly wanted to determine the PVRIG RNAexpression profile in cyno PBMC subsets. As no cPVRIG primers setexisted, we designed two sets that were directed at two distinct siteson the cPVRIG gene. One primer set was specific for the X2 variant ofcPVRIG, while the other set was able to pick up both the X1 and X2variant. As shown in FIG. 35, both primer sets were able to detectcPVRIG RNA at a similar level when compared to each other. Furthermore,unlike human PBMC where there was a distinct PVRIG RNA signature ineffector lymphocytes (NK and T cells) compared to monocytes, cPVRIG RNAwas expressed at a similar level across all PBMC subsets from all donorsassessed.

PVRIG protein expression on cynomolgus PBMC is very low or negative:Having established a cPVRIG RNA profile for cyno PBMC, we screened forthe presence of cPVRIG protein on cyno PBMC using a select panel ofanti-human PVRIG antibodies. The antibodies chosen to screen PBMC werebased on their ability to bind cPVRIG transient cells and/or functionalactivity. As shown in FIGS. 36A-36C, we were able to detect low level ofexpression of cPVRIG on the CD16+ lymphocyte subset (NK cells) from arange of antibodies, but not the CD3+ lymphocyte subset (T cells) northe CD14+CD56+ myeloid subset (monocytes). Despite this data, thoseantibodies that showed positive detection over control (as denoted bythe solid black line) did not correlate to those that were able to bindthe cPVRIG transient cells. For example, the level of staining byCPA.7.021 was more than CPA.7.024 despite the former not binding tocPVRIG transient cells (see FIGS. 36A-36C).

Summary and Conclusions: Using an antibody phage platform, we have beenable to successfully generate monoclonal antibodies towards the humanPVRIG antigen. Using engineered over-expressing cells as well as a suiteof cancer cell lines, we showed that our antibodies are highly specificto the PVRIG antigen, and are able to detect protein expression whichcorrelated with RNA expression. Upon analysis of human PBMC subsets, weshowed that the PVRIG protein is most highly expressed on NK cells, withlow expression on conventional CD3+ T cells, and not detectable on Bcells and myeloid cells. The expression did not robustly change uponexposing these cell types to various stimulation conditions. We alsoshowed that a panel of our antibodies are cross-reactive with thecynomolgus monkey (cyno) PVRIG antigen through assessing their bindingto over-expressing cells. However, the combination of the low level ofbinding of this panel of antibodies to cyno PBMC, the lack of proteincorrelation with RNA, and the discordance of their ability to bind toover-expressing cells (compared to PBMC) indicates that the PVRIGantigen on cyno PBMC may be very low/negative, or it is expressed in adifferent/more complex form compared to the over-expressing cells.

Example 1D

Expression of PVRIG in PBMC subsets from healthy donors: The expressionof PVRIG in PBMC subsets from healthy donors was tested (gating strategyis shown in FIG. 1a ). In the tested samples, PVRIG was shown to expresson CD8+ T cells (data not shown), CD8α+γδ T cell (data not shown),double-negative γδ T cells (data not shown) and to a milder extent alsoon CD4+ T cells (data not shown) of healthy donors PBMCs (n=5).

Example 1E

Co-expression of PVRIG with PD1. TIGIT and HLA-DR in Ovarian Cancerascites, PBLs of MSS, CRC, and in resting and allo-activated healthyPBMCs: PVRIG is co-expressed with TIGIT on CD8+ T cells in ovariancancer ascites (data not shown). In this sample, a mixed level of PVRIGexpression was observed, that overlapped with that of PD-1 expression.Low level of HLA-DR correlated with low level of PVRIG expression. Verylow level of PVRIG was observed on CD4+ T cells is in this specificsample, indicating no correlation with PD1, TIGIT and HLA-DR.

In PBLs of MSS CRC patients, PVRIG is co-expressed with TIGIT on CD8+ Tcells (data not shown). Low expression levels of PVRIG were observed inthis sample which was in correlation with the low levels of TIGIT andHLA-DR. TILs from this patient had small CD8+ population that stainedpositive for surface PVRIG, which was also positive for PD1 and TIGIT(data not shown). Intracellular stain reveled prominent PVRIG stain thatmirrored the expression pattern of PD-1, showing two distinctpopulations that are PD1-PVRIG- and PD1+ PVRIG+(data not shown).Intracellular PVRIG+CD8+ T cells seem to better correlate with theHLA-DR+ and TIGIT+. PVRIG was not detectable on the surface of CD4+ Tcells and only minority of the CD4+ cells showed positive intracellularPVRIG stain in the PD1+ population. Due to the very small intracellularPVRIG+ population, it is difficult to determine if PVRIG is co-expressedwith TIGIT and HLA-DR.

In healthy PBMCs, PVRIG stain on CD8 T cells mirrored the expressionpattern of PD-1 and TIGIT, showing distinct PD1-PVRIG- and PD1+ PVRIG+populations and distinct TIGIT-PVRIG- and TIGIT+ PVRIG+ populations(data not shown). PVRIG was not detected on CD4+ cells. Interestingly,following allo-activation, co-expression of PVRIG and PD-1 was observedon CD4+(but no on CD8+) (data not shown).

In summary, PVRIG was shown to co-express with TIGIT in CD8+ T cellsfrom ovarian cancer ascites, MSS CRC patient's PBLs and with PD-1healthy donor's PBMCs and with PD1 in CD4+ T cells of allo activatedPBMCs from healthy donor.

Example 1F

Expression of PVRIG on lymphocyte populations from Healthy PBMCs Urachalcancer, colorectal cancer, ovarian cancer ascites and lung cancer:Results: The expression of PVRIG on CD4+ and CD8+ T cells, NK cells andon CD4+ and CD8+NKT cells was analyzed in healthy donors' PBMCs andtonsils and in TILs from urachal cancer, colorectal cancer, ovariancancer ascites, lung cancer and melanoma.

In healthy donors' PBMCs (n=5) and in ovarian cancer ascites TILs (n=1)high levels of PVRIG expression was detected on NK cells (data notshown) and CD8+NKT cells (data not shown) and to a lower extent also onCD8+ T cells (data not shown) and CD4+NKT (data not shown). CD4+ T cellsalso stained positively for PVRIG in some of the PBMCs, however thelevel of expression was quite low (data not shown).

In addition, PVRIG expression was detected on CD4+ T cells from two outof 6 colorectal cancer TILs tested, and in lung cancer TILs (n=3) (datanot shown) and on NK cells from urachal cancer TILs (n=1).

No PVRIG expression was detected in melanoma TILs due to absence of TILsin the tested sample.

Example 1G

Additional evaluations were done to identify addition tissues that overexpress PVRIG in human and mouse cell lines.

Reagents: Human PVRIG TaqMan probes (Life technologies) Hs04189293_g1,Cat. #4331182, TaqMan probe for Housekeeping gene (HSKG) (Lifetechnologies) human RPL19 Mm 01577060_gH, human HPRT1 Hs02800695_ml,human SDHA Hs00417200_ml, human PBGD Hs00609296_g1, and human TATA BoxHs00375874_g1. Mouse PVRIG TaqMan probes (Life technologies) CC70L8H,CC6RN19 Custom TaqMan probes. TaqMan probes for Housekeeping gene (HSKG)(Life technologies) mouse RPL19: Mm02601633_g1. ABI TaqMan Fast AdvancedMaster mix, part no. 4444557, Applied Biosystem. Commercial Human andMouse cancer cell lines from American Type Culture Collection (ATCC) andCLS (Cell line service) are detailed in Table 1. RNA extraction fromhuman and mouse cell lines was performed with RNAeasy Mini Kit (Qiagencat #74014). cDNA was produced using High Capacity cDNA ReverseTranscription Kit (Applied Biosystems cat #4368814. Commercial mousepolyclonal Anti-PVRIG Ab MaxPab (BO), Abnova, Cat #H00079037-B01,diluted 1:200. Mouse IgG1, Life Technologies, Cat #MG100, diluted 1:200.Commercial mouse polyclonal Anti-PVRIG Ab, Sigma, Cat #SAB1407935, 10μg/ml. Chrom pure Mouse IgG, whole molecule, Jackson, Cat #015-000-003,10 μg/ml. Goat Anti Mouse-PE, Jackson, Cat #115-116-146, diluted 1:100.Custom polyclonal Rat-Anti mouse PVRIG, Batch #20153456C.1, Aldevron, 10μg/ml. Custom Rat total IgG, Batch #GV20884.1, Aldevron, 10 μg/ml. GoatAnti Rat-PE, Jackson, cat #112-116-143, diluted 1:100. Anti-humanPVRIG-CPA.7.024 mIgG1 conjugated to AF647, 10 μg/ml. Anti-humanPVRIG-CPA.7.050 mIgG1 conjugated to AF647, 10 μg/ml. Anti-humanPVRIG-CPA.7.005 mIgG1 conjugated to AF647, 10 μg/ml. Anti-humanPVRIG-CPA.7.002 mIgG1 conjugated to AF647, 10 μg/ml. Synagis IgG1conjugated to A647, 10 μg/ml. Anti-human PVRIG-CPA.7.021 mIgG1conjugated to AF647, 10 μg/ml. Synagis IgG2 conjugated to A647, 10μg/ml. Rabbit polyclonal anti PVRIG Ab, Sigma, Cat #HPA047497, diluted1:300. Goat Anti Rabbit-HRP, Jackson, Cat #111-035-003, diluted 1:100.VioBlue, Fixable viability stain 450, BD Bioscience, cat #562247,diluted 1:1000. Human Trustain FcX, Biolegend, Cat #422302. Rat antimouse CD16/CD32 Fc block, BD, Cat #553142. Ingenio Electroporationsolution, Mirus, Cat #MIR50114. ON-TARGETplus Human PVRIGsiRNA-SMARTpool, Dharmacon, Cat # L-032703-02. ON TARGET plus nontargeting siRNA, Dharmacon, Cat # D-001810-01-05. The human cell linesused in the study are shown in FIG. 54.

Transcript expression. Quantitative RT-PCR (qRT-PCR): RNA (1-5 ug)extraction of human and mouse cell lines (detailed above in Tables 1 and2) was preformed according to manufactures protocols. cDNA was preparedaccording to manufactures protocols (1 ug RNA diluted in 20 ul cDNA mixreaction). cDNA, prepared as described above, diluted 1:10 (representing25 ng RNA per reaction), was used as a template for qRT-PCR reactions,using a gene specific TaqMan probes (detailed in materials & methods 1.11-4) Detection was performed using QuantStudio 12 k device. The cycle inwhich the reactions achieved a threshold level of fluorescence(Ct=Threshold Cycle) was registered and was used to calculate therelative transcript quantity in the RT reactions. The absolute quantitywas calculated by using the equation Q=2{circumflex over ( )}−Ct. Theresulting relative quantities were normalized to a relative quantitiesof housekeeping gene, mRPL19 or hRPL19.

Protein expression detection by Western Blot (WB): The expression ofhuman PVRIG in human cell lines was analyzed by WB using whole cellextracts (45 ug for the cancer cell lines, and 30 ug for the overexpressing cell line and negative control cell line). Commercial rabbitpolyclonal anti-human PVRIG pAb, Sigma, cat # HPA047497, diluted 1:300in 5% BSA/TBST followed by secondary Ab goat anti-Rabbit-Peroxidaseconjugated (Jackson, cat #111-035-003), diluted 1:20,000 in 5% milkTBST.

Protein expression analysis by Flow Cytometry (FACS): The cell surfaceexpression of PVRIG protein was analyzed by FACS. Human or mouse celllines were stained with VioBlue reagent diluted 1:1000 in PBS. Cellswere incubated 15 min at R.T. and then washed once with PBS. Cell linesfor endogenous protein analysis were pre-incubated with the Fc receptorblocking solutions listed above in material section (2.5 μl/reaction ofhuman blocker and 1 μl/reaction of mouse blocker was used according tothe manufactures procedures). To detect the human PVRIG protein, cellswere stained with a commercial polyclonal anti human PVRIG or by acustom monoclonal anti-human PVRIG mAbs (Inc production, detailed inmaterials & methods section above) diluted to a concentration of 10μg/ml or 1:200 (for Sigma Ab and for mAb or for Abnova Ab respectively)or IgG1 Isotype control at the same concentration followed by Goat antimouse PE conjugated Ab.

To detect the mouse PVRIG protein, cells were stained with a Custom ratpolyclonal anti-mouse PVRIG pAb (Aldevron) diluted to a concentration of10 μg/ml or rat IgG whole molecule as isotypes control at the sameconcentration followed by Donkey anti Rat-PE conjugated Ab diluted1:100.

PVRIG knock down: Knock down of endogenous human PVRIG was carried outby transient transfection of siRNA. Transfection of 100 pmol PVRIG siRNApool or scrambled siRNA performed by electroporation using Amaxanucleofector device and MIRUS Ingenio electroporation solution, aslisted above in materials & methods and according to the manufactureprocedure. 48 hours post transfection, cells were collected for furtheranalysis by qRT-PCR and FACS.

Results: Endogenous expression of the PVRIG transcript in human andmouse cell lines by qRT-PCR

Human cell lines: In order to verify the presence of the PVRIGtranscript in human cell lines (listed in FIG. 54), qRT-PCR wasperformed using a specific TaqMan probe as describe above in Material &Methods. As shown in FIGS. 56A-56C. human PVRIG transcript is observedusing TaqMan probe Hs04189293_g1 with relatively high levels in Jurkat(A, B), HUT78 (A, B) and HL60 (B) cell lines. Lower transcript level isobserved in THP1, RPM18226 (B) cell lines. All other cell lines showvery low to no transcript.

Endogenous expression of the PVRIG transcript in mouse cell lines byqRT-PCR: In order to verify the presence of the PVRIG transcript inmouse cell lines (listed in FIG. 55), qRT-PCR was performed using aspecific TaqMan probe as describe above in Material & Methods. As shownin FIGS. 57A-57B mouse PVRIG transcript is observed using TaqMan probeCC70L8H with relatively high levels in NIH/3T3, Renca, SaI/N and J774A.1(A), cell lines. Lower transcript level is observed in CT26 (A) andB-104-1-1(B) cell lines. All other cell lines show very low transcript.

Endogenous expression of the PVRIG proteins in human cell lines by WB:WB analysis for endogenous expression of PVRIG protein was carried outon various human cancer cell lines lysates as detailed in FIG. 54 usingcommercial anti human PVRIG pAb (Sigma, HPA047497) as described inMaterials & Methods above. As a positive control, whole cell extract ofstable HEK293 cell pool over-expressing PVRIG was used while cellstransfected with an empty vector served as the negative control. Asshown in FIG. 58, a protein band corresponding to ˜35 kD was detected inthe positive control HEK293 over expressing cells (lane 2), as well asin the Jurkat cell line (lane 3). No expression of human PVRIG wasdetected in the empty vector cells (lane 1) which served as a negativecontrol nor in ZR75-1 human cell line (lane 4).

Endogenous Expression of the PVRIG Proteins in Human and Mouse CellLines by FACS:

Human cell line: To verify the cell-surface endogenous expression ofhuman PVRIG, various human cell lines (detailed in FIG. 54) were testedas described in Material & Methods above. The cell lines were stainedwith the commercial Ab (Abnova) or with Isotype control followed by asecondary goat anti mouse PE Ab. Analysis was performed by FACS. Bindingof Abnova antibody was observed in Jurkat human cancer cell line ascompared to isotype control binding. No binding of Abnova Ab wasobserved in the other tested cell lines: For Capan2 and ZR75-1 ascompared to isotype control binding, additional FACS analysis was doneusing Sigma commercial Ab on a various human cell lines (Jurkat, HUT78,Karpas299 and NK-YTS), binding was observed in Jurkat cells only but nobinding was observed to other cell lines (data not shown).

Further analysis for endogenous confirmation of human PVRIG in Jurkatcell line, was done by testing binding of various monoclonal antibodiesof the invention. Jurkat cell line was stained with five anti-humanPVRIG custom mAbs (CPA.7.024, CPA.7.050, CPA.7.005, CPA.7.002 andCPA.7.021) conjugated to AF647 or with relative Isotype control Abconjugated to AF647 Analysis was performed by FACS. The expression ofhuman PVRIG in Jurkat human cell line was observed by CPA.7.021 andCPA.7.050 only, as compared to isotype control expression. No bindingfor human PVRIG was observed in Jurkat cell line by using the otherthree mAbs.

Mouse cell line: to verify the cell-surface endogenous expression ofmouse PVRIG, various mouse cell lines: J774A.1, NIH/3T3, SaI/N and Renca(detailed in FIG. 55), were tested as described in Material & Methodsabove. The cell lines were stained with the custom polyclonal rat antimouse PVRIG Ab (Aldevron), or with Isotype control (Aldevron) followedby a secondary goat anti rat PE Ab. Analysis was performed by FACS. Nobinding for mouse PVRIG protein was observed in either of the testedmouse cell lines by Aldevron polyclonal Ab (data not shown).

Knock down of human PVRIG in human cell lines: In order to furtherconfirm endogenous expression of PVRIG protein in Jurkat cell line,human PVRIG siRNA pool was used for knock down as described in Material& Methods. 48 hours post siRNA transfection, cells were harvested forfurther analysis by qRT-PCR and by FACS.

Knock down of human PVRIG in human cell lines tested by qPCR: As shownin FIG. 59 human PVRIG transcript level in Jurkat cells transfected withhuman PVRIG siRNA pool is significantly reduced (right histogram bar) ascompared to cells transfected with scrambled siRNA (left histogram bar)analyzed by qRT-PCR as described in Material & Methods.

Knock down of human PVRIG in human cell lines tested by FACS: Furtheranalysis of human PVRIG membrane expression in the same siRNAtransfected cells was performed by FACS. As shown in FIG. 60 membraneexpressions of human PVRIG protein is reduced in cells transfected withPVRIG siRNA (green for CPA.7.021 mAb or red for Sigma Ab) as compared tocells transfected with scrambled siRNA (orange). The fold change (antiPVRIG vs, Isotype control) in Jurkat cell line is decreased from 8 foldto 3.3 fold by using Sigma Ab, or from 15.3 fold to 2.8 fold by usingCPA.7.021 mAb.

This report includes preliminary data on PVRIG endogenous expression incell lines both at the RNA level and the protein level in human andmouse cell lines.

Various human cancer cell lines were tested by qRT-PCR, WB and FACS forendogenous expression of PVRIG.

Cell surface expression of human PVRIG was observed in Jurkat cell lineby using the commercial polyclonal Abs (Sigma and Abnova) and the mousemonoclonal Abs (Inc), as shown in FIGS. 4A and 4B respectively. Theseobservations are in correlation to RNA transcript levels as shown inFIGS. 1A & B, and to WB results as shown in FIG. 3.

Additional confirmation of endogenous human PVRIG in Jurkat cell lineswas done by knock down experiment confirming clear reduction in the RNAtranscript following PVRIG siRNA transfection, as shown in FIGS. 5A-5C,and also reduction was observed in the protein cell surface expressionin Jurkat cell lines as shown in FIG. 6 by commercial Ab and bymonoclonal Ab.

Various mouse cell lines were tested by qRT-PCR and FACS for endogenousexpression of PVRIG. In the transcript level, presence of PVRIG wasobserved in J774A.1, NIH/3T3, SaI/N and Renca cell lines as shown inFIGS. 2A & B. Although no membrane expression of mouse PVRIG wasobserved in these tested cell lines detected by polyclonal Ab (Aldevron)(data not shown). FIG. 61 and FIG. 62 indicate the summary of thefindings described in this report, highlighting the cell lines showingcorrelation between qPCR and FACS, confirmed by knock down.

Example 1H

The aim of this experiments is to evaluate the expression of PVRIGprotein on resting or activated human (Tumor infiltrating lymphocytes)TILs isolated from human melanoma samples and propagated in the presenceof melanoma specific antigens and IL2. Human mAb were produced directedagainst the extracellular domain (ECD) of human PVRIG. These Abs weredirectly labeled with Alexa flour 647 in order to examine the expressionof PVRIG on cells by FACS analysis.

Materials and Methods

TILs: In this experiments series three different Tumor-infiltratinglymphocyte (TIL) from resected metastases of three melanoma patientswere used: 1) TIL-412-HLA-A2-Mart1 specific; 2) TIL-F4-HLA-A2-gp100specific, and 3) TIL-209-HLA-A2-gp100 specific. Human TILs (>90% CD8+),were thawed 24h prior to beginning of experiment. Cells were thawed in12 ml of TIL medium (IMDM+10% human serum+1% Glutamax+1% Na-Pyruvate+1%non-essential amino acids+1% Pen-Strep) supplemented with 300 U/ml ofrhIL2 (Biolegend 509129). Cells were left to recover from freezing for24 hours.

Assay conditions: After recovery, TILs were tested in four differentconditions: 1) Resting—with 300 U/ml of IL2 (Biolegend cat-589106), 2)With polyclonal activation of T cells, using 1 μg/ml of plate bound antiCD3 antibody (eBioscience clone OKT3, cat-16-0037-85)+2 μg/ml of antiCD28 ab (eBioscience clone CD28.2 cat-16-0289-85)+300 U/ml of IL2, 3)Co-cultured (1:1) with Mel888 (LIMS ID: CL-216) melanoma cells (HLA-A2negative) and 4) Co-cultured (1:1) with Mel624 (LIMS ID CL-218) melanomacells (HLA-A2+Mart1/gp100 positive).

After 12 hours of resting/activation/co-culture, cells were tested byFACS for PVRIG expression as well as the expression of other members ofPVRIG pathway and other surface markers.

Staining cells: Cells were harvested after 12 hours and washed twicewith PBS. Cells were stained in room temp for 20 minutes with PBSsupplemented with 1/1000 of fixable viability stain efluor 450 (BDhorizon cat-562247). After staining, cells were washed twice with PBSand stained for 15 minutes on ice with FACS buffer (PBS+0.5% BSA+2 mMEDTA+0.05% Azide) supplemented with 1/25 of human Truestain FC-Block(Biolegend, 422302). After FC-blocking, cells were stained on ice for 30minutes with the Abs and concentrations that are listed in table 1.

Conjugated Catalog concentration Staining Antibodies Isotype toManufacturer number (ug/ul) concentration Anti-human Human AF-647Compugen - CPA.7.021 0.2 5 μg/ml PVRIG - IgG2 iNC CPA.7.021 Human HumanAF-647 Compugen - 0.2 5 μg/ml IgG2 IgG2 iNC isotype control CD96 mIgG1APC Biolegend 338410 0.2 4 μg/ml PVR mIgG1 APC Biolegend 337618 0.05 1μg/ml PVRL2 mIgG1 APC Biolegend 337412 0.1 2 μg/ml TIGIT mIgG1 APCeBioscience 17-9500-42 0.025 0.5 μg/ml DNAM1 mIgG1 APC Biolegend 3383120.1 2 μg/ml PD1 mIgG1 AF647 Biolegend 329910 0.1 2 μg/ml CD8 mIgG1 FITCBiolegend 300906 0.15 3 μg/ml

After staining, cells were washed once and re-suspended in FACS bufferfor analysis. Compensation calibration was done using compensation beads(BD, 552843). One drop of beads were stained for 30 minutes with aboveantibodies. Beads staining was done with same concentrations as cellstaining. After beads staining, compensation was performed on MacsQuantFACS machine according to standard procedure. All samples were acquiredon a MACSQuant analyzer (Miltenyi) and data was analyzed using Tree StarFlowJo software (v10.0.8).

PVRIG is expressed on human resting TILs: Resting TILs, cultured for 12hours with 300 U/ml of IL2 only, were stained for PVRIG expression andanalyzed by FACS. Gating strategy for TILs: Lymphocytes were gated firstaccording to size and granularity in FCS:SSC graph, than single cellswere gated according to FSC-H and FSC-A, than live cells were gatedaccording to viability Dye staining in Vioblue:FSC graph, than CD8+cells were gated according to CD8 staining in CD8:FSC graph. Expressionlevels of PVRIG was than plotted according to PVRIG staining inhistograms.

PVRIG expression on human TILs is downregulated upon activation withanti CD3+anti CD28 abs: Human TILs, cultured for 12 hours with antiCD3+anti CD28 abs+IL2 were stained for PVRIG expression and analyzed byFACS. PVRIG expression on surface of all three TILs examined isdownregulated upon activation, comparing to resting TILs (data notshown).

PVRIG expression on human TILs is slightly downregulated upon co-culturewith Mel888: Human TILs, co-cultured for 12 hours with Mel888 cells werestained for PVRIG expression and analyzed by FACS. PVRIG expression onsurface of all three TILs examined is slightly downregulated uponco-culture with Mel888 comparing to resting TILs.

PVRIG expression on human TILs is downregulated upon co-culture withMel624: Human TILs, co-cultured for 12 hours with Mel624 cells werestained for PVRIG expression and analyzed by FACS.PVRIG expression onsurface of all three TILs examined is slightly downregulated uponco-culture with Mel624 comparing to resting TILs.

Expression of other pathway members on resting TILs: Human TILs,co-cultured for 12 hours with IL2 only were stained for the expressionof CD96, PVR, PVRL2, TIGIT and DNAM1 and analyzed by FACS. CD96, TIGITand DNAM1 is expressed on all three examined TILs. PVR is expressed onthe surface of all three TILs as well but to relatively low levels.PVRL2 is not detected on any of the TILs.

Expression of other pathway members on TILs activated with anti CD3 andanti CD28 abs: Human TILs, cultured for 12 hours with anti CD3 and antiCD28 abs were stained for the expression of CD96, PVR, PVRL2, TIGIT andDNAM1 and analyzed by FACS. Upon activation with anti CD3+anti CD28 abs,CD96 is downregulated, PVR is slightly upregulated, TIGIT is slightlyupregulated and DNAM1 is upregulated as well.

Expression of other pathway members on TILs Co-cultured with Mel888:Human TILs, co-cultured for 12 hours with Mel888 cells were stained forthe expression of CD96, PVR, PVRL2, TIGIT and DNAM1 and analyzed byFACS. Upon co-culture with Me888, CD96 is downregulated, PVR is highlyupregulated, TIGIT and DNAM1 is downregulated, PVRL2 is slightly inducedas well.

Expression of other pathway members on TILs Co-cultured with Mel624:Human TILs, co-cultured for 12 hours with Mel624 cells were stained forthe expression of CD96, PVR, PVRL2, TIGIT and DNAM1 and analyzed byFACS. Gating strategy was done according to FIG. 1. Upon co-culture withMel624, CD96 is downregulated, PVR is highly upregulated, TIGIT isstable or slightly upregulated, DNAM1 is downregulated and PVRL2 isslightly induced.

Expression of PD1 on TILs: Human TILs, cultured for 12 hours with IL2only or activated with anti CD3+anti CD28 abs or co-cultured with Mel888or with Me624 cells were stained for the expression of PD1 and analyzedby FACS. As can be seen in FIG. 16 and FIG. 17, PD1 is expressed onresting TIL412 only. No change in PD1 expression is noticed uponco-culture with Mel888, But, PD1 is upregulated in all three TILs uponco-culture with Mel624 or upon activation with anti CD3+anti CD28 abs.

Summary and conclusions: For all TILs that were tested:

-   -   Anti PVRIG—CPA.7.021 ab stains TILs (up to 2.6 fold)    -   PVRIG expression is downregulated upon activation of 12 hours        with anti CD3+anti CD28 abs or upon co-culture with Me624        (almost to background level).    -   Resting TILs express CD96, TIGIT and DNAM1 (up to 35, 12 and 79        fold respectively)    -   CD96 expression is downregulated upon activation (from up to 35        to ˜11 fold) or co-culture with irrelevant (HLA-A2-) melanoma    -   DNAM1 expression is upregulated upon activation with αCD3/CD28        abs (from up to 79 to 102 fold) but strongly downregulated upon        co-culture of TILs with Mels (down to 8 fold).    -   TIGIT expression is slightly downregulated upon co-culture of        TILs with me888 cell line, and was stable with a slight        upregulation upon co-culture with Mel624 or activation with anti        CD3+anti CD28 abs.    -   PD1 expression is upregulated upon activation (from 0 up to 18        fold)    -   High levels of PVR were detected following TILs co-culture with        melanomas (from <2 up to 18 fold).

Resting TIL-412 show positive staining for PD1. TIL-F4 is also slightlypositive for PD1 whereas TIL-209 is negative. Summary of changes inexpression levels of all parameters tested, in the different conditionscan be seen in Table 2.

TABLE 2 +IL2 +αCD3+αCD28+IL2 +Mel888 +Mel624 PVRIG 1.4-2.6  0-12 1.3-1.7  0-1.2 CD96 23-35 12.7-16   11.7-17.6 11.1-16.6 TIGIT  5.7-12.6 7.8-12.5   4-7.3  6.1-12.5 DNAM1 43-79  56-100 14-20 17-25 PVR 1.6-1.82.6-3.2 13.6-18   11-17 PVRL2 0 0 1.4-2.3 1.2-1.8 PD1   0-4.5  2.3-18.4  0-4.6   2-9.3

Example 11: Expression of PVRIG on Resting and Activated Human T Cellsand TILs

The aim of this example was to evaluate the expression of PVRIG proteinon resting and activated human isolated primary CD4+ and CD8+ T cells,as well as TILs (Tumor Infiltrating Lymphocytes) isolated from humanmelanoma samples and propagated in the presence of melanoma specificantigens and IL2. Human mAbs were produced against the extracellulardomain (ECD) of human PVRIG. These Abs were directly labeled with Alexaflour 647 in order to examine the expression of PVRIG on cells by FACSanalysis.

Materials and Methods

TILs: In this series of experiments, two different TILs, from resectedmetastases of three melanoma patients, were used:

TIL-Mart1-HLA-A2-Mart1 specific

TIL-209-HLA-A2-gp100 specific

Human TILs (>95% CD8+), were thawed 24h prior to beginning ofexperiment. Cells were thawed in 12 ml of TIL medium (IMDM+10% humanserum+1% Glutamax+1% Na-Pyruvate+1% non-essential amino acids+1%Pen-Strep) supplemented with 300 U/ml of rhIL2 (Biolegend 509129). Cellswere left to recover for 24 hours.

Primary T cell: In this series of experiments two different donors wereused:

CD4+ and CD8+ from donor #147

CD4+ and CD8+ from donor #186

Human primary cells (>95% purity), were thawed 24h prior to beginning ofexperiment. Cells were thawed in RPMI complete medium (RPMI+10% FBS+1%Glutamax+1% Na-Pyruvate+1% Pen-Strep) supplemented with 300 U/ml ofrhIL2 (Biolegend 509129). Cells were left to recover for 24 hours.

Assay conditions: After recovery, cells were activated using apolyclonal activation of T cells, with 1 μg/ml of plate bound anti CD3antibody (BD-pharmingen clone Ucht-1, cat-555329), 2 μg/ml of anti CD28ab (eBioscience clone CD28.2 cat-16-0289-85) and 300 U/ml of IL2.

Activation was carried out for 24h, 48h, 72h and 144h.

Staining cells: Cells were harvested and washed with PBS. Cells werestained at room temprature for 10 minutes with PBS supplemented with1/1000 of fixable viability stain efluor 450 (BD horizon cat-562247).After staining, cells were washed twice with PBS and stained with theAbs at the concentrations listed in

FIG. 65 for 30 minutes on ice in FACS buffer (PBS+0.5% BSA+2 mMEDTA+0.05% Azide) and concentrations that are listed in

FIG. 65. After staining, cells were washed once and re-suspended in FACSbuffer for analysis.

Results: Human T cells from two different donors and TILs were leftuntreated (resting) or polyclonal stimulated for various timepoints asdescribed in Materials and Methods. Cell activation state was evaluatedby detection of surface expression of CD137 and PD-1 at each time pointcompared to isotype control (FMO), as shown for activated CD8+, CD4+ Tcells and TILs (FIGS. 70A, B & C respectively). As expected, PD-1 andCD137 expression was detected and elevated upon activation (FIGS. 70A, B& C).

PVRIG expression was observed on both resting CD4+ and CD8+ T cells,with higher expression on CD8+ cells (6-8 fold) as compared to CD4+cells (3 folds), and diminished upon activation (FIGS. 71A, B & C). Ondays 3-6 of activation, PVRIG expression was increased on CD8+(4-5 fold)and CD4+(2-3 fold) T cells, as can be seen in FIGS. 71A-71C.

In addition, PVRIG expression was also observed on Mart1 and 209 restingTILs, and expression was decreased apon activation (FIGS. 721A, B & C).On day 3-6 of activation PVRIG expression was increased, as can be seenin FIG. 72, compared to day 1-2 of activation.

Example 2: Generation and Characterization of PVRIG-Expressing StableTransfectant Cell Pools

Recombinant stable pools of cell lines overexpressing PVRIG human andmouse proteins were generated, for use in determining the effects ofPVRIG on immunity, for PVRIG characterization and for identifyingimmunoregulatory PVRIG based therapeutic agents.

Materials & Methods:

Reagents: DNA constructs:

Human PVRIG flag pUC57

Human PVRIG flag pCDNA3.1

Human PVRIG flag pMSCV

Recombinant cells:

HEK293 pCDNA3.1 Human PVRIG flag

HEK293 pMSCV Human PVRIG flag

Commercial antibodies:

Anti PVRIG, Sigma cat. HPA047497—Rabbit polyclonal

Anti-PVRIG, Abnova cat. H00079037-B01-Mouse polyclonal

Full length validation of mouse PVRIG was done using PCR reactions andsequencing of the PCR products.

Three couples of primers were used (Table 3).

TABLE 3 Sequence of primers used for mouse full length validationPrimer name Sequence: 200-554_mPVRIG_F CCACCAACCTCTCGTCTTTC(SEQ ID NO: 1531) 200-553_mPVRIG_R TCATGCCAGAGCATACAG (SEQ ID NO: 1532)200-571_mPVRIG_F CAGTGCCTCTAACTGCTGAC (SEQ ID NO: 1533) 200-572_mPVRIG_RTCACTGTTACCAGGGAGATGAG (SEQ ID NO: 1534) 200-549_mPVRIG_FCACAGGCTGCCCATGCAAC (SEQ ID NO: 1535) 200-551_mPVRIG_RTGCCTGGGTGCTAGTGAGAG (SEQ ID NO: 1536) 200-554_mPVRIG_FCCACCAACCTCTCGTCTTTC (SEQ ID NO: 1537) 200-546_mPVRIG_RGACCCTGTTACCTGTCATTG (SEQ ID NO: 1538)

As a templet for the PCR reaction, cDNA of NIH 3T3 cell line or a mix ofthree commercial cDNA panels were used:

1. cDNA panel I, Mouse, Biochain, Cat no. C8334501 (Heart, Brain,Kidney, Liver).

2. cDNA panel II, Mouse, Biochain, Cat no. C8334502 (Lung, Pancreas,Spleen, Skeletal Muscle).

3. cDNA, Clontech, Cat no. 637301, (Brain, Heart, day 7 Embrio, Testis,Spleen).

Expression Constructs

Full length cloning of human and mouse PVRIG-flag was performed by genesynthesis (GenScript) using codon optimized sequence in pUC57 vector forhuman transcript and non optimized for mouse transcript and subclonedinto a mammalian expression vector, pcDNA3.1 or to pMSCV, to create theexpression plasmid.

Human PVRIG sequence that was subcloned into pcDNA3.1 initiate from thesecond methionine of human PVRIG protein, whereas the human PVRIGsequence that was subcloned into pMSCV initiate from the firstmethionine of human PVRIG protein.

-   -   Construct encoding the Human PVRIG-flag.

Full length human PVRIG gene, synthesis by GenScript was subcloned intousing pcDNA3.1 using BamI and NheI restriction enzymes.

Constructs encoding the mouse PVRIG proteins:

Four contracts encoding the mouse sequence were synthesize by GenScriptas following:

1. First Methionine no tag

2. First Methionine with Flag

3. Second Methionine no tag

4. Second Methionine with Flag

The synthesize gene were subcloned into pCDNA3.1

Generation of stable transfectants over expressing PVRIG proteins

The resulting expression construct was verified by sequence andsubsequently used for transfections and stable pool generation asdescribed below. The protein sequences encoded by the expressionconstructs are as set forth in FIGS. 103A-103BX.

Generation of stable transfectant pools expressing human PVRIG-flagprotein

HEK293 (ATCC, catalog number: CRL-1573) cells were transfected withpCDNA3.1+ human PVRIG-flag plasmid or with empty vector (pCDNA3.1+ asnegative control), using FUGENE 6 Reagent (Roch, catalog number11-988-387). Geneticin, G418 (Gibco, catalog number: 11811-031)resistant colonies were selected for stable pool generation.

GP2-293 packaging cell line (Clontech cat #631458) was transfected withpMSCV-human PVRIG or with pMSCV empty vector using Lipofectamine 2000transfection reagent (Invitrogen, catalog number 11668019). 48 hourspost transfection supernatants containing virions were collected, anddirectly used for infection of the human cell line as follows:

HEK-293 (ATCC, CRL-CRL-1573) cells was infected with virions expressinghuman PVRIG or with pMSCV empty vector virions as negative control,Puromycin (Invivogen, catalog number: 58-58-2) resistant colonies wereselected for stable pool generation.

Expression Validation

Expression Validation by Western Blot

Whole cell extracts of cell pool (30 ug of total protein) were analyzedby western blot. As negative control, whole cell extracts of stable cellpools transfected with the empty vector were used. For the humanPVRIG-flag detection, anti-flag and anti PVRIG antibodies were used asfollow:

-   -   Mouse anti Flag M2-Peroxidase, Sigma, cat. A8592 diluted 1:1000        in TTBS/5% BSA;    -   Anti PVRIG, Sigma cat. HPA047497—Rabbit polyclonal, diluted        1:200 in TTBS/5% BSA. Followed by Goat Anti Rabbit-HRP, Jackson,        Cat: 111-035-003 diluted 1:20,000 in 5% milk/TTBS

solution.

Expression validation by Flow Cytometry (FACS)

In order to validate the cell surface expression of the human PVRIGprotein in the recombinant stable pools, 1×10⁵ cells were stained withFixable viability stain 450 (BD, 562247) diluted 1:1000 in PBS, for 10min at R.T. Mouse polyclonal anti PVRIG, (Abnova, Cat.H00079037-B01)diluted 1:200 or with mouse IgG1 isotype control (Life Technologies),were then added to cells followed by staining with Goat Anti Mouse-PE(Jackson, cat.115-116-146).

Results Expression Validation of HEK293 Stable Pool Cells OverExpressing the Human PVRIG-Flag Protein

To verify expression of the PVRIG protein in the stably transfectedHEK293 cells pools, whole cell extracts were analyzed by western blotusing anti-flag antibody or anti PVRIG antibodies (Abnova), as describedin Material and Methods. The results, shown in FIG. 24, demonstrate aband corresponding to the expected protein size of ˜33 kDa in theextracts of HEK293 cell pools expressing human PVRIG, but not in thecells transfected with the empty vector.

In order to verify cell surface expression of the PVRIG protein, HEK293stably transfected cells over-expressing the PVRIG-flag pCDNA3.1 vectorwere analyzed by FACS using mouse anti-PVRIG pAb (Abnova) as describedin Material and Methods. The results presented in FIG. 25 show that thebinding of mouse anti-PVRIG pAb to cells stably expressing the humanPVRIG-flag (gray) is higher than that observed with cells transfectedwith the empty vector (light gray).

Example 3: PVRIG-ECD Ig Fusion Protein Production

PVRIG mECD-mIg fusion protein (see FIGS. 103A-103BX), composed of theECD of mouse PVRIG fused to the Fc of mouse IgG2a, was produced atProBioGen (Germany) in CHO-DG44 cells by culturing stable cell pools for12 days, followed by Protein A purification of cell harvest andpreparative SEC purification for aggregate removal. The final productwas formulated in 5 mM Na citrate, 5 mM Na/K phosphate, 140 mM NaCl,0.01% Tween pH5.5.

Expression vector used was ProBioGen's PBG-GPEX6. PVRIG gene is drivenby CMV/EF1 hybrid promoter followed by polyadenylation signal pA-1. Thevector contains puromycin N-acetyl-transferase gene that allowsselection of transfected cells using puromycin, as well as dehydrofolatereductase gene that allows selection of transfected cells usingmethotrexate (MTX).

PVRIG hECD-hIg fusion protein (see FIGS. 103A-103BX), composed of theECD of human PVRIG fused to the Fc of human IgG1 bearing C220, C226 andC229 to S mutations at the hinge, was produced at GenScript (China) bytransient transfection in CHO-3E7 cells which were cultured for 6 days,followed by protein A purification of cell harvest. The final productwas formulated in PBS pH 7.2.

Expression vector used was Mammalian Expression Vector pTT5, in whichPVRIG gene is driven by CMV promoter.

Example 4: Expression of PVRIG on Human PBLs and Binding of PVRIG-Fc toMelanoma Cell Lines

PVRIG is a novel immune checkpoint protein, which without wishing to belimited by a single theory functions as a CD28 like receptor on T cells.In this study, the expression of PVRIG on human peripheral bloodlymphocytes and the binding of PVRIG-ECD-Ig (composed of theextra-cellular domain of human PVRIG fused to human IgG1) to melanomacell lines was evaluated.

Materials and Methods

Three human melanoma cell lines which present the MART-1 antigen inHLA-A2 context (SK-MEL-23, Mel-624 and Mel-624.38) were used as targetsfor CTLs. Mel-888 which does not express HLA-A2, served as a negativecontrol.

Buffy coats from human healthy donors were obtained from Tel HashomerBlood Bank. Peripheral blood mononuclear cells were stimulated with PHAand cultured for 3 days, and subsequently transduced with MSCV-basedretroviral vector (pMSGV1). Following transduction, cells were furthergrown in lymphocyte medium (Bio target medium, fetal bovine serum (10%),L Glutamine Penicillin/Streptomicyn (100 units/ml), IL-2 300 IU) foradditional 5 days.

To evaluate PVRIG expression on PBLs, cells were stained with a specificantibody for PVRIG (mouse poly clonal) at 5 μg/ml for 30 min at 4degrees. Following washing, cells were stained with FITC conjugated Goatanti mouse mAb (1:250) (Invitrogen, Cat # A10667) in FACS buffer in thedark for 30 minutes at 4 degrees. Following two washes in FACS buffer,samples were read on a BD Bioscience FACS Calibur with a Cytek HTS.

To evaluate binding of PVRIG-Ig to the melanoma cell lines, SK-MEL-23,Mel-624, Mel-624.38 and mel-888, cells were co-cultured with F4transduced or un-transduced (designated w/o) PBLs and subsequentlystained with 20 μg/ml of the fusion protein PVRIG-Ig HH batch #125.Following two washes in FACS buffer, samples were stained with asecondary goat anti-human PE (Jackson, cat #109-116-098).

Results

To evaluate the endogenous expression of PVRIG on primary humanleukocytes, PBLs were stimulated with PHA and subsequently transducedwith an empty vector and stained with an anti-PVRIG specific antibody.As shown in FIG. 11, in two different donors staining with anti-PVRIG isobserved relative to an isotype matched control.

To evaluate the endogenous expression of PVRIG on melanoma cell linesand to determine whether the endogenous expression is affected byco-culture with antigen specific T cells, 4 different melanoma celllines (SK-MEL-23, Mel-624, Mel-624.38 and mel-888) cu-cultured with PBLseither expressing or not expressing the F4 (gp100 specific TCR). Cellswere subsequently stained with the fusion protein composed of theextra-cellular domain of human PVRIG fused the Fc portion of human IgG1.As shown in FIG. 12, all 4 tested human melanoma cell lines exhibitbinding to PVRIG-Ig. Binding intensity is not affected by T celldependent activation following co-culture with melanoma reactiveengineered T cells.

Summary: The results presented herein suggest that PVRIG is expressed onPHA activated human primary peripheral blood leukocytes (PBLs). Inaddition, 4 melanoma cell lines that were tested in this study bind tothe fusion protein composed of the extra-cellular domain of human PVRIGfused the Fc portion of human IgG1 suggesting that these cell linesexpress the counterpart for PVRIG.

Example 5: Receptor Ligand Identification and Validation

A first validation study was performed using a cell microarraytechnology was used to screen for interactions of PVRIG to 3559full-length human plasma membrane proteins, which were individuallyexpressed in human HEK293 cells.

Human HEK293 cells were grown over slides spotted with expressionvectors encoding 3559 full-length human membrane proteins. An expressionvector (pIRES-hEGFR-IRES-ZsGreen1) was spotted in quadruplicate on everyslide, and was used to ensure that a minimal threshold of transfectionefficiency had been achieved or exceeded on every slide. Human HEK293cells were used for reverse transfection/expression. A fusion proteincomposed of the ECD of PVRIG fused to a human IgG1 was added at 20 μg/mlto each slide following cell fixation. Detection of binding wasperformed by using an appropriate fluorescent secondary antibody. Tworeplicate slide-sets were screened. Fluorescent images were analyzed andquantitated (for transfection efficiency) using ImageQuant software(GE).

A protein ‘hit’ was defined as a duplicate spot showing a raised signalcompared to background levels. This was achieved by visual inspectionusing the images gridded on the ImageQuant software. Hits wereclassified as ‘strong, medium, weak or very weak’, depending on theintensity of the duplicate spots. To confirm the hits, all vectorsencoding the hits identified in the primary screen were arrayed on newslides. Confirmation/Specificity screen and analyses was carried out asfor primary screening (n=2 replicate slides per sample), except thatidentical slides were also probed with appropriate negative controls.Additionally, all the vectors encoding the hits were sequenced. Vectorsencoding every primary hit was sequenced confirming its identity.

Background screen showed negligible binding to untransfected HEK293cells at 2, 5 and 20 μg/ml (FIG. 13). Based upon the background data, 20μg/ml was chosen for full profiling. Primary screen resulted in multipleduplicate hits (clones), with the majority being weak or very weakintensity. All primary hits identified, and a control EGFR-ZsGreen1vector, were spotted and re-expressed in duplicate and probed with PVRIGat 20 μg/ml for the Confirmation/Specificity screen.

A single specific hit, PVRL2, with strong intensity, was identified(FIG. 14). Another weak hit, MAG, was later shown to bind also otherfusion proteins tested (data not shown), thus suggesting that it is notspecific. These results are consistent with the recently publishedabstracthttps://www.yumpu.com/en/document/view/7263720/sunday-december-4-late-abstracts-1-molecular-biology-of-the-/133by G. Quinones in New Technologies & Frontlers. PVRL2 is known to play arole as a ligand for TIGIT and DNAM1, which are both modulators of Tcell and NK cell activation. TIGIT has been recently reported to be akey player in the inhibition of the immune response directed againsttumor cells (Noa Stanietsky, journal of immunology, vol. 106 no. 42,17858-17863; Robert J Johnston, Cancer cell, Volume 26, Issue 6, p923-937, 8 Dec. 2014). Results presented in Example 5, showinginteraction of PVRIG with the same counterpart as TIGIT, suggests aninvolvement of PVRIG in an important regulatory pathway that regulatescancer immune surveillance and thus positions PVRIG as a potentialtarget for cancer treatment.

Additional Validation Study 2

Materials and Methods

Materials

Fc fusion proteins, His-tagged proteins and control Ig: The Fc fusionprotein PVRIG-Fc M:M was used for binding studies. Mouse IgG2a was usedas isotype control. Other commercial mouse proteins used in the studywere PVRL2-his (R&D, 3869-N2), and PVRL2-his (Sino Biological,50318-M08H).

Cells: HEK293 over-expressing (OX) mouse PVRIG and PVRIG-FLAG weregenerated (RC-287 and RC-286, respectively) and binding of PVRL2 tothese cells was compared to HEK293 cells expressing empty vector (EV)(RC-83). HEK293 OX mouse PVRL2 splice variants 1 and 2 (sv1 and sv2)were generated (RC-334 and RC-335, respectively) and binding of PVRIG tothese cells was compared to HEK293 cells expressing EV. B16-F10 cells(CL-161, mouse skin melanoma cells endogenously expressing mPVRL2) werealso used to study the interaction between PVRIG and PVRL2.

Antibodies: Anti-mouse PVRL2-PE Ab (R&D, FAB3869P, 25 μg/ml, 1:100) wasused for detection of PVRL2. Rat IgG2A-PE (R&D, IC006P, 25 μg/ml, 1:100)was used as isotype control. Anti-mouse-PE (Jackson Immunoresearch,115-115-206, 0.5 mg/ml, 1:200) and anti-his Ab (Abcam, ab72467, 0.1mg/ml, 1:300) were used to detect binding of recombinant proteins.Anti-DYKDDDDK Tag (“DYKDDDDK” disclosed as SEQ ID NO: 6) (anti-FLAG) Ab(BioLegend, 637302, 0.5 mg/ml, 1:300) was used for detection of PVRIGexpression on HEK293 OX mouse PVRIG-FLAG.For PVRIG labeling, AlexaFluor® 647 Antibody Labeling Kit (Molecular Probes, A-20186) was usedaccording to manufacturer's protocol. For biotinylation of PVRIG, DSB-X™Biotin Protein Labeling Kit (Molecular Probes, D-20655) was usedaccording to manufacturer's protocol. Biotinylated PVRIG was detected bystreptavidin-PE (SA-PE) (Jackson Immunoresearch, 016-110-084, 0.5 mg/ml,1:300).

Methods

FACS analysis of mouse PVRIG-Fc binding to stable HEK293 cellsover-expressing (OX) mouse PVRL2 or to B16-F10 cells: HEK293 cells OXPVRL2 (sv1 or sv2) or B16-F10 cells were suspended to 10⁶ cells/ml inPBS. For each 1 ml of cells, 1 μl of viability stain stock solution (BDHorizon Fixable Viability Stain 450, cat. 562247, BD Bioscience) wasadded. Cells were incubated for 10 min protected from light at roomtemperature. The cells were then washed twice with PBS and suspended to3×10⁶ cells/ml in the presence of 1:50 human TruStain FcX™ (BioLegend422302) in FACS buffer (PBS supplemented with 2% FBS and 0.5 mM EDTA) atroom temperature for 15 min for blocking of Fcγ-receptors. Withoutwashing, 1×10⁵ cells/well were then plated in 96-well V-shaped plates(Costar #3357). Expression of PVRL2 was examined by anti-PVRL2 antibody(see above). Binding of PVRIG-Fc to cells was examined with variousbatches (see above), generally at 60 μg/ml or with severalconcentrations. Cells were incubated with antibodies or PVRIG-Fc for 40min at room temperature, then washed once. Secondary antibody(anti-mouse-PE) was added for 15 min at room temperature, cells werewashed twice and were taken for analysis by MACSQuant® FACS analyzers(Miltenyi Biotec), followed by data analysis using Flow-Jo 10 software.

FACS analysis of mouse PVRL2-his binding to stable HEK293 cells OX mousePVRIG: PVRIG levels were examined with anti-FLAG antibody. PVRL2-hisbinding was monitored by anti-his antibody. FACS analysis was performedas described above.

Biophysical SPR analysis of mouse PVRIG/PVRL2 interaction by Biacore:The interaction between mouse PVRIG and PVRL2 was analyzed in a BiacoreT100 SPR biomolecular interaction analyzer at Bar-Ilan University.Proteins were diluted to 100 nM in acetate buffer pH 4.0, and werecovalently coupled to a unique flow cell of a CM5 Series S Biacore chipusing standard amine coupling chemistry. Surfaces were activated withEDC-NHS, and later blocked by injection of 1M ethanolamine (pH 8.5).Running buffer was 10 mM Hepes pH 7.3, 150 mM NaCl, 3 mM EDTA and 0.05%Tween-20 (HBS-EP+). Final immobilization levels were ˜1000 RU. Proteinsused as analytes were diluted to 2500 nM, 500 nM and 100 nM. In each runone tube contained running buffer only for reference. After each run aregeneration step with 4M MgCl2 for 30 sec at 20 μl/sec was performed.

Results

Binding of mouse PVRIG to HEK293 cells OX PVRL2 sv1: In order tovalidate the interaction between mouse PVRIG and mouse PVRL2 we firsttested the binding of PVRIG-Fc to cells over-expressing (OX) PVRL2. Thelevel of PVRL2 expression on HEK293 OX PVRL2 sv1 was determined usingspecific anti-mouse PVRL2 antibodies. Mouse PVRL2 expression was 10-foldhigher compared to HEK293 cells expressing empty vector (data notshown). Four batches of PVRIG-Fc were examined for binding to PVRL2 OXcells. All PVRIG-Fc batches showed 6-11-fold binding to cells OX PVRL2compared to empty vector cells (data not shown). Binding of PVRIG-Fc toPVRL2 OX cells was also examined using biotinylated and fluorescentlylabelled (Alexa Fluor 647) PVRIG proteins. While the biotinylatedproteins displayed slightly stronger binding to PVRL2 OX cells comparedto untagged PVRIG-Fc (data not shown), fluorescently labelled PVRIGdemonstrated much lower binding (data not shown). These results showthat PVLR2 is detected on the membrane of HEK293 cells OC PVRL2; bindingof mouse PVRIG-Fc to PVLR2 OX cells is detected by anti-mouse IgG2Aantibodies; binding of biotinylated mouse PVRIG-Fc to PVLR2 OX cells isdetected by streptavidin-PE, and binding of Alexa Fluor 647-labeledPVRIG-Fc to PVLR2 OX cells.

Binding of mouse PVRL2 to HEK293 cells OX PVRIG: To further validate theinteraction between mouse PVRIG and mouse PVRL2 we tested the binding ofPVRL2 to cells OX PVRIG with or without a FLAG-tag. Membrane expressionof mouse PVRIG on HEK293 cells OX PVRIG with a FLAG-tag was confirmedusing an anti-FLAG antibody (data not shown). As expected, HEK293 cellsOX PVRIG without a FLAG-tag showed no expression using an anti-FLAGantibody. Using anti-PVRIG supernatants (Aldeveron), these cellsdemonstrated lower expression of PVRIG compared to cells OX PVRIG with aFLAG-tag. Commercial mouse PVRL2 recombinant protein was available onlyas a His-tagged protein. Therefore, extensive calibrations were requiredto obtain an appropriate anti-His antibody and conditions for detection.His-tagged PVRL2, from two different sources, were tested for binding toPVRIG OX cells at 60 μg/ml and demonstrated 2-fold (data not shown) and3-4 fold (data not shown) binding compared to HEK293 cells expressingempty vector. That is, his-tagged mouse PVLR2 binds HEK293 OX mousePVRIG, and mouse PVRIG is expressed on membranes of HEK293 cells OXPVRIG.

Study of mouse PVRIG and mouse PVRL2 interaction using SPR-Biacore: Inorder to assess the interaction between mouse PVRIG-Fc and mouseHis-tagged PVRL2, both proteins were immobilized to a Biacore chip.Following immobilization, both proteins, as well as PVRIG-Fc (data notshown) were run as analytes at three concentrations: 2500, 500 and 100nM (PVRIG batch #480 and PVRL2 were run twice as analytes). Interactionbetween the two proteins was detected in both directions and with bothbatches of PVRIG (data not shown). Due to complex kinetics, an exact KDcould not be determined from the Biacore results.

Dose response binding of mouse PVRIG to HEK293 cells OX PVRL2 sv2 andB16-F10 cells: As shown above, mouse PVRL2 binding to mouse PVRIG OXcells was relatively low. In order to establish a method for screeninganti-mouse PVRIG antibodies capable of blocking the interaction betweenmouse PVRIG and mouse PVRL2, the binding of PVRIG-Fc to PVRL2 OX cellswas selected. First, a dose response binding curve of mouse IgG2A andmouse PVRIG-Fc to cells OX mouse PVRL2 was generated and compared tocells expressing empty vector (EV). The dose response was performed intwo-fold serial dilutions (1:2) from 50 μg/ml to 0.1p g/ml. While nodifference in mouse IgG2A binding was observed (data not shown),PVRIG-Fc demonstrated saturation of binding at 12.5 μg/ml and reducedbinding in correlation with the decrease in protein concentration (datanot shown). Similar results were obtained also with PVRIG-Fc (data notshown). These results suggest that this binding assay can be consideredfor screening of blocking antibodies.

In order to consider also an endogenous system for screening ofanti-mouse PVRIG antibodies, the expression of PVRL2 on B16-F10 cellswas assessed using an anti-PVRL2 antibody. Results show that PVRL2 ishighly expressed on B16-F10 cells (data not shown). Therefore, a similardose response binding curve was produced also for binding of mouse IgG2Aand mouse PVRIG-Fc to B16-F10 cells. Similarly to the results obtainedwith HEK293 cells OX PVRL2, mouse PVRIG-Fc demonstrated dose responsebinding to B16-F10 cells reaching saturation at 12.5 μg/ml, while nochange in binding of mouse IgG2A was detected (data not shown).

Discussion and Conclusions: Human PVRIG interaction with human PVRL2 wasidentified using Cell Microarray Technology at Retrogenix. To validatethis interaction also in mouse, several approaches were taken. Amongthem the use of PVRIG or PVRL2 OX cells, and biophysical measurementsusing SPR-Biacore. All approaches indicated that mouse PVRIG interactswith mouse PVRL2. However, the binding of mouse PVRL2 to cells OX PVRIGwas relatively low compared to the binding of PVRIG to cells OX PVRL2.The reason for this could be the fact that commercial PVRL2 is availableonly as a monomer His-tagged protein and not as an Fc-fused protein (asfor PVRIG). To this end, a custom Fc-fused mouse PVRL2 was produced atGenScript. However, from preliminary data, only a minor increase inbinding was observed with this protein (˜5-fold compared to 2-3 foldwith the PVRL2-his). Therefore, some other factors might influence thisrelatively low binding.

Due to the low PVRL2 binding to cells OX PVRIG, it was decided toestablish an anti-PVRIG antibody blocking assay using PVRIG-Fc bindingto cells OX PVRL2. According to the observed dose response curves wesuggested three working concentrations: 0.1, 0.2 and 0.4 μg/ml.Following similar results obtained with binding of PVRIG to PVRL2endogenously expressing B16-F10 cells, we suggested to perform theantibody blocking assay also on these cells at the followingconcentrations: 0.2, 0.4, 0.8 μg/ml.

PVRIG is a presumed receptor, therefore, preferably the antibodyblocking assay should be performed with PVRL2 as a soluble protein andPVRIG expressed on the cells. Thus, it should be considered to examineanti-mouse PVRIG antibodies that demonstrate blocking activity in thecurrent format also in this system.

Additional Validation Study 3

The objective of this study is to confirm the binding partners of PVRIG,a novel immuno-oncology target. Preliminary studies indicate that one ofthese ligands is PVRL2. In this study, binding of the recombinant PVRIGprotein to several potential ligands in the PVRIG axis has beeninvestigated by ELISA.

Protocols

List of reagents: Current literature on the PVRIG proteins suggests thatthere are three potential ligands: PVR (CD155), PVRL2 (CD112), and PVRL3(CD113). To investigate their ability to bind the PVRIG receptor, thesethree ligands were sourced commercially, as follows: PVR and PVRL3 fromSino Biologicals Inc. and PVRL2 from R&D Systems and Sino BiologicalsInc. The human PVRIG recombinant protein was generated at Compugen asthe PVRIG extra-cellular domain (ECD) fused to a human IgG1 Fc domain(PVRIGHH).

ELISA to determine receptor-ligand interaction: Commercially sourcedHis-tagged ligands, PVR, PVRL2, and PVRL3, were coated on the wells of ahigh binding EIA/RIA plate (Costar 9018) overnight at 4° C. Anirrelevant His-tagged protein was included as a negative control. Coatedplate wells were rinsed twice with PBS and incubated with 300 μLblocking buffer (5% skim milk powder in PBS pH 7.4) at room temperature(RT) for 1 hr. Blocking buffer was removed and plates were rinsed twicemore with PBS. Plate-bound ligands were incubated with varyingconcentrations of PVRIGHH in solution (linear range of 0.1 μg/mL to 4μg/mL in a 50 μL/well volume) at RT for 1 hr. Plates were washed threetimes with PBS-T (PBS 7.4, 0.05% Tween20), then three times with PBS and50 μL/well of a HRP-conjugated secondary antibody was added (Human IgGFc domain specific, Jackson ImmunoResearch). This was incubated at RTfor 1 hr and plates were washed again. ELISA signals were developed inall wells by adding 50 μL of Sureblue TMB substrate (KPL Inc) andincubating for 5-20 mins. The HRP reaction was stopped by adding 50 μL2N H2SO4 (VWR) and absorbance signals at 450 nm were read on aSpectraMax (Molecular Devices) or EnVision (PerkinElmer)spectrophotometer. The data were exported to Excel (Microsoft) andplotted in GraphPad Prism (GraphPad Software, Inc.).

Results: PVRIG preferably binds to PVRL2: The human PVRIG Fc-fusionprotein was assayed for binding to PVR, PVRL2 and PVRL3, which wereimmobilized on an EIA/RIA plate. Varying concentrations of the receptorPVRIG in solution phase were incubated with the immobilized ligand. Thedata clearly show dose-dependent binding of PVRIGHH to PVRL2, but nobinding to ligands PVR, PVRL3 or the negative control protein (data notshown). The ELISA A450 signal was plotted as a function of the receptorconcentration using a one-site binding equation, revealing anequilibrium binding constant (KD) of 13±1 nM.

Summary and Conclusions: PVRIG is a novel immuno-oncology target forwhich the biology is not fully understood. In an effort to shed morelight on this biology, we examined its binding to several potentialligands. PVRL2 was clearly identified as the binding partner of PVRIG.Quantitative analysis suggests that this interaction is very strong,with a KD of 131 nM. Our results also suggest that human PVRIG eitherdoes not bind the human PVR and PVRL3, or the binding is too weak todetect by ELISA.

Additional Validation Study 4:

In this example, PVRIG expression on PBMC cell subsets was evaluated preand post allo-activation. Following allo-activation the expression ofPVRIG was upregulated on CD4+ T cells as well as on CD8+ T cells anddouble negative gamma delta T cells. This upregulation was observed inPBMCs of one out of two donors tested (see FIGS. 52A-52B).

Example 6 Surface Plasmon Resonance Studies of PVR, PVRL2, and PVRL3Binding to PVRIG, DNAM, And TIGIT

Materials and Methods

All experiments were performed using a ProteOn XPR 36 instrument at 22°C.

Step 1: A high density goat anti-human fc polyclonal antibody surface(Invitrogen H10500) was prepared over all six lanes of a GLC chip usinga ProteOn XPR 36 biosensor. The activation step for the anti-human fcsurface occurred in the horizontal flow direction while theimmobilization step for the high density pAb occurred in the verticalflow direction. The blocking step occurred in both the vertical andhorizontal positions so that the horizontal “interspots” could be usedas reference surfaces. An average of ˜4400 RU of goat anti-human pAb wasimmobilized on each lane.

Step 2: For each cycle, three different lots of human PVRIG fusionprotein (human fc, GenScript lots 451, 448, 125), human DNAM-1 fusionprotein (human fc, R&D Systems), human TIGIT fusion protein (human fc,R&D Systems), and a control human IgG (Synagis) were each captured overa different vertical lane for two minutes at a concentration of 2 μg/mL.PVR, two lots of PVRL2, and PVRL3 were each injected in the horizontalflow direction at six different concentrations over all six capturedligands at different ligand capture cycles. The injections were twominutes followed by 10 minutes of dissociation at a flow rate of50p/min. The PVR concentration range was 1.4 nM-332 nM in a 3-folddilution series, both lots of PVRL2 were injected at a concentrationrange of 1.3 nM-322 nM in a 3-fold dilution series, and PVRL3 wasinjected at a concentration range of 1.4 nM-334 nM in a 3-fold dilutionseries. All protein reagents were prepared in running buffer which wasdegassed PBS buffer with 0.05% Tween 20 and 0.01% BSA added. Theanti-human fc capture surfaces were regenerated with two 30-secondpulses of 146 mM phosphoric acid after each cycle.

Step 3: Sensorgram data of the analytes binding to each captured ligandwere processed and double-referenced using ProteOn Manager version3.1.0.6 making use of interspot referencing and a pre-blank injectionidentical to the analyte injections.

Results

a) PVR: Binds weakly to captured DNAM-1 and TIGIT and shows no bindingto all three lots of PVRIG and the control IgG. Not enough informationwas generated to estimate the KD of the PVR interactions with DNAM-1 andTIGIT (data not shown).

b) PVRL2: Both lots of PVRL2 showed binding to all three lots of PVRIGand to DNAM-1 but minimal or no binding to TIGIT and no binding to thecontrol IgG. Sensorgrams showed complex kinetics, therefore bindingconstants could not be estimated (data not shown).

c) PVRL3: Showed minimal binding to TIGIT and did not bind the otherproteins (data not shown).

Example 7: In-Vitro Immunomodulatory Activities of PVRIG ECD-IG on MouseT Cells

In these experiments the immunomodulatory activities of the recombinantfused protein PVRIG-ECD-Ig was investigated on mouse T cell activation.The effect of PVRIG-ECD-Ig on activation of mouse CD4 T cells wasinvestigated using a number of in-vitro T cell activation readouts: cellactivation markers, cytokine secretion and proliferation.

In order to evaluate the activity of pvrig protein on t cell activation,recombinant protein was produced comprising the mouse extracellulardomain (ECD) of the mouse PVRIG fused to the Fc of mouse IgG2a(designated PVRIG-ECD IG M:M) (SEQ ID NO:29). The effect of the Fc fusedprotein co-immobilized with anti-CD3 on mouse CD4 T cell functions, asmanifested by activation markers and cytokines secretion wasinvestigated.

Materials and Methods

Fc fusion protein and control Ig: Fc fusion protein, PVRIG-ECD-Ig (batch#198) was tested. Mouse IgG2a (clone MOPC-173; Biolegend or C1.18.4;BioXcell) was used as isotype control.

Mouse CD4 T cells isolation: Untouched CD4+CD25− T cells were isolatedfrom pools of spleens of BALB/C mice using a T cell isolation Kit(Miltenyi Cat #130-093-227) according to the manufacturer'sinstructions. The purity obtained was >90%.

Activation of mouse CD4 T cells: Anti-mouse CD3-E mAb (clone 145-2C11;BD Biosciences) at 2 μg/ml together with PVRIG-ECD-Ig protein or controlIg at various concentrations (1, 3 or 10 μg/ml), were co-immobilized for3 hr at 37° C., on 96-well flat bottom tissue culture plates (Sigma,Cat. # Z707910). Control Ig was added to each well in order to completea total protein concentration of 12 μg/ml per well. Wells were washed 3times with PBS and plated with 1×10⁵ purified CD4+CD25− T cells per welland kept in a humidified, 5% CO2, 37° C. incubator. In some experiments,soluble anti-CD28 (clone: 37.51; eBioscience; 1 μg/ml) was added.Culture supernatants were collected at the indicated times poststimulation and analyzed for mouse IFNγ or IL-2 secretion by ELISA kits(R&D Systems). The effect of PVRIG-ECD-Ig protein (see FIG. 103) on theexpression of the activation marker CD69 on mouse CD4+ T cells wasanalyzed by flow cytometry. Cells were stained 48h post stimulation witha cocktail of antibodies including PerCP-anti-CD4 (clone G41.5;Biolegend), FITC or PE-anti-CD69 (clone H1.2F3; Biolegend), in thepresence of anti-CD16/32 (clone 2.4 g2; BD Biosciences) for blocking ofFcγ-receptors. Cells were evaluated using MACSQuant analyzer 9(Miltenyi) and data analyzed using BD CellQuest or by MACSQuantify™Software. Data was analyzed using Excel or Prism4 software.

Results and Summary

Effect of PVRIG-ECD Ig M:M (see FIG. 103) on mouse CD4+ T cellsfunction: FIGS. 15A-15E show in-vitro immunomodulatory activities ofPVRIG-ECD-Ig (see FIG. 103) on isolated mouse splenic T cells(CD4+, >95% purity) stimulated with microplates co-immobilized withanti-CD3 (2 μg/ml) alone or co-immobilized with control Ig (mIgG2a) orPVRIG-ECD-Ig (see FIG. 103)) (10 μg/ml) in the presence of solubleanti-CD28 (1 μg/ml). PVRIG-ECD-Ig (see FIG. 103) suppressed mouse CD4 Tcell activation in a dose dependent manner, as manifested by reducedCD69 up-regulation (FIGS. 15A, D), and reduction in TCR-inducedcytokines (IL-2 and IFNγ) secretion (FIGS. 15B-C, E). The magnitude ofthe inhibitory effect of PVRIG-ECD-Ig ((see FIG. 103)) was in the rangeof 30-100%. Inhibitory effect of PVRIG-ECD-Ig ((see FIG. 103)) on IFNγsecretion was observed in concentrations as low as 3 μg/ml (60%inhibition vs. control Ig).

PVRIG-ECD-Ig (see FIG. 103) inhibits T cell activation in aconcentration-dependent manner when the Fc fusion protein isco-immobilized with anti-CD3 on plates. Maximal inhibitory effect wasobserved at 10 μg/ml of PVRIG-ECD-Ig (see FIG. 103).

The results demonstrate the inhibitory effect of PVRIG-ECD-Ig on mouse Tcells activation, manifested by reduced cytokine secretion, andsuppression of activation marker CD69 upregulation. This inhibition of Tcell activation, supports the therapeutic potential of immunoinhibitoryPVRIG proteins (PVRIG polypeptides and fusion proteins) according to thepresent invention in treating T cell-driven autoimmune diseases, such asrheumatoid arthritis, multiple sclerosis, psoriasis and inflammatorybowel disease, as well as for other immune related diseases and/or forreducing the undesirable immune activation that follows gene therapy. Inaddition, these results also support the therapeutic potential ofimmunostimulatory PVRIG proteins (PVRIG polypeptides and fusionproteins) that reduce the inhibitory activity of PVRIG for treatingconditions which should benefit from enhanced immune responses, inparticular enhanced CTL immunity and proinflammatory cytokines such ascancer, infectious diseases, particularly chronic infections and sepsiswherein T cell-mediated depletion of diseased cells is therapeuticallyadvantageous.

Example 8: In-Vitro Immunomodulatory Activities of PVRIG on HumanCytotoxic T Cells (CTLs)

The experiments described in this example evaluated the effect ofectopic expression of human PVRIG on different melanoma cell lines ontheir ability to activate CTLs (cytotoxic T lymphocytes) and serve astargets for killing by these cells.

Materials & Methods:

Three human melanoma cell lines which present the MART-1 antigen inHLA-A2 context (SK-MEL-23, Mel-624 and Mel-624.38) were used as targetsfor CTLs. Mel-888 which does not express HLA-A2, served as a negativecontrol.

Ectopic expression of human PVRIG on cytotoxic T lymphocytes (CTLs): Inorder to express human PVRIG in peripheral blood leukocyte (PBL)cultures, the cDNA encoding for PVRIG was amplified using specificprimers and cloned into an MSCV-based retroviral vector (pMSGV1) or intripartite vectors: the CD8-dependent F4 TCR α- and β-chains were linkedwith a P2A sequence and cloned into pMSGV1 vector, either followed by aninternal ribosome entry site (IRES) and PVRIG. The retroviral vectorencoding for NGFR1, as negative control or in tripartite vectors: theCD8-dependent F4 TCR α- and β-chains were linked with a P2A sequence andcloned into pMSGV1 vector, either followed by an internal ribosome entrysite (IRES) and NGFR Verification of the cloning was done first usingrestriction enzyme digestion and subsequently by sequencing. Uponsequence confirmation, large amounts of the retroviral vector(Maxi-prep) were produced for subsequent use.

Peripheral blood leukocytes of healthy human donors were transduced withthe retroviral constructs encoding PVRIG or with the retroviral vectorsencoding for NGFR1 or an empty vector, as negative control. Transductionwas carried out using a retronectin-based protocol; briefly, retroviralsupernatant was produced in 293GP cells (a retroviral packaging cellline) following transfection with the retroviral vector and anamphotropic envelop gene (VSV-G). The retroviral supernatant was platedon retronectin-coated plates prior to the transduction to enable thebinding of virions to the plate, and the PBLs were added to the platefor 6 hours. After that, the cells were replenished in a new culturevessel. Transduction efficiency and expression of the protein wasdetermined by staining the transduced PBLs with commercial PVRIGspecific rabbit polyclonal antibody or with commercial anti-NGFR (Cat.No345108; BioLegend). Rabbit IgG (Sigma Cat. No. 15006) was used asisotype control, and as secondary antibody we used APC-conjugatedanti-rabbit IgG (Jackson, Cat. No. 711-136-152).

Ectopic expression of the F4 T cell receptor on cytotoxic T lymphocytes(CTLs): In order to obtain effector lymphocytes that express theMART-1-specific F4 TCR, specifically recognizing MART-126-35-/HLA-A2peptide-MHC complex, freshly isolated human PBLs previously transducedto express either with PVRIG, NGFR or an empty vector were stimulatedwith PHA and cultured for 5-10 days, and subsequently transduced with invitro-transcribed mRNA encoding both α and β chains from theMART-1-specific F4 TCR. The transduced lymphocytes were cultured inlymphocyte medium (Bio target medium, fetal bovine serum (10%), LGlutamine Penicillin/Streptomicyn (100 units/ml), IL-2 300 IU),replenished every 2-3 days. F4 TCR expression levels were verified byFACS staining using a specific monoclonal antibody that recognizes theextra-cellular domain of the beta-chain from the transduced specificTCR. (TCR-Vb12-PE, (Cat.No IM2291; Beckman Coulter).

Cytokine secretion from PVRIG, NGFR or an empty vector and F4-TCRtransduced lymphocytes upon co-culture with melanoma cells: PBLsexpressing PVRIG or NGFR along with F4-TCR were co-cultured withun-manipulated melanoma cells. 105 transduced PBLs were co-cultured with105 melanoma target cells for 16 hours. In order to assess the responseof the effector CD8 T cells to the different tumor cell lines, cytokinesecretion (IFNγ, IL-2 and TNF-α) was measured by ELISA in culturesupernatants (IFNγ (Cat.No DY285E), IL-2 (Cat.No DY202E), TNF-α (Cat.NoDY210E) R&D SYSTEMS), diluted to be in the linear range of the ELISAassay.

Cell mediated cytotoxicity assay: This assay was performed in order toasses target cell killing upon co-culture. PVRIG and F4 were expressedin PBLs using a bi-cystronic vector and co-cultured with CFSE labeledmelanoma Target cells (labeled with 2 mM CFSE (eBioscience) for 6 min),at 37° C. for 18 hr, at E:T ratio of 3:1. Cells were collected after 18hr and and 1 mM propidium iodide (Sigma-Aldrich) was added for assigningthe ratio of cell death. Samples were run on a CyAn-ADP flow cytometer(Beckman Coulter).

Results:

General design of the experimental system: In the experimental systemdescribed herein, PVRIG is over expressed on human PBLs which are nextmanipulated to express the MART1-specific and HLA-A2 restricted F4 TCR.Over expressing cells are then co-cultured with HLA-A2 positive (namethem) and HLA-A2 negative (names) melanoma cell lines (reference). TheF4 TCR was recently used in clinical trials in terminally-ill melanomapatients to specifically confer tumor recognition by autologouslymphocytes from peripheral blood by using a retrovirus encoding the TCR(Morgan et al, 2006 Science, 314:126-129). The effect of PVRIGexpression on antigen-specific activation of CD8 T cells by co-culturewith cognate melanoma cells was assessed by cytokine secretion.

Over expression of PVRIG on human PBLs—experiment 1: Human PBLs weretransduced with a retroviral vector encoding the PVRIG or an emptyvector as negative control, as described in Materials & Methods. Thelevels of PVRIG were assessed by flow cytometry at 48 hrs aftertransduction, and compared to cells transduced with an empty vector. Thepercentage of the transgene-expressing cells was 62.4% as shown in FIG.16.

Over expression of PVRIG on human PBLs—experiment 2: Human PBLs weretransduced with a retroviral vector encoding the PVRIG or NGFR or anempty vector as negative controls, as described in Materials & Methods.The levels of PVRIG were assessed by flow cytometry at 48 hrs aftertransduction, and compared to cells transduced with an empty vector. Thepercentage of the PVRIG-expressing cells was in the range of 20%. Theexpression of NGFR was of 63% as shown in FIG. 17. A few additionalattempts to over express PVRIG on PBLs were un-successful. Onepossibility is that the difficulty in expressing PVRIG in primary PBLsstems from a basal endogenous expression level in these cells.

Over expression of F4 TCR on human PBLs: To perform functional assayswith human CTLs, we used PBLs engineered to express the F4 TCR, whichrecognizes HLA-A2+/MART1+ melanoma cells, as described in Materials &Methods. FIG. 18A shows levels of F4 TCR expression obtained upon TCRtransduction of leukocytes used in experiment 1, FIG. 18B shows levelsof F4 TCR expression obtained upon TCR transduction of leukocytes usedin experiment 2.

Effect of PVRIG expression on IFNγ secretion—experiment 1: PVRIG orEmpty-vector and F4-transduced PBLs were co-cultured with melanoma celllines. The levels of IFNγ secretion were measured at 16-hours ofco-culture. As shown in FIG. 19, the magnitude of inhibition of IFNγsecretion due to PVRIG over-expression was more than 90%. Co-culturewith the HLA-A2 negative cell line Mel-888 which served as a negativecontrol, caused only a minor activation dependent IFNγ secretion fromF4-transduced lymphocytes. PBLs not expressing the F4 TCR (designatedW/O) serve as an additional negative control.

Effect of PVRIG expression on IFNγ secretion—experiment 2: PVRIG, NGFRor Empty-vector and F4 were transduced into PBLs in co-transduction(FIG. 20A) or using a bi-cystronic vector (FIG. 20B). Transduced PBLswere co-cultured with melanoma cell lines. The levels of IFNγ secretionwere measured at 16-hours of co-culture. As shown in FIG. 20A, themagnitude of inhibition of cytokine secretion due to PVRIGover-expression was in the range of 30%. Co-culture with the HLA-A2negative cell line Mel-888 which served as a negative control, causedonly a minor activation dependent IFNγ secretion from F4-transducedlymphocytes. PBLs not expressing the F4 TCR (designated W/O) serve as anadditional negative control. As shown in FIG. 20B, when PVRIG isco-transduced with the F4 TCR, no inhibition of IFNγ was observed.

Effect of PVRIG on CTL mediated killing activity—experiment 2: PVRIG orNGFR and F4 were transduced to PBLs using a bi-cystronic vector andco-cultured with CFSE labeled melanoma cell lines. As shown in FIG. 21,the percentage of propidium Iodide positive events (reflecting intensityof killing activity) was decreased by 50% by the expression of PVRIGrelative to negative control NGFR transduced cells. Killing activity ofPVRIG expressing cells is similar to that of co-culture between melanomaand PBLs not expressing the F4 TCR (designated W/O).

Summary: Without wishing to be limited by a single hypothesis, theresults presented herein indicate that overexpression on primarylymphocytes results in reduced cytokine secretion by CTLs, suggestingthat PVRIG has an inhibitory effect on CTLs.

Example 9: Human Anti-PVRIG Antibodies

The objective of this study was to isolate human antibodies that bind tothe PVRIG immuno-oncology target with high affinity and specificity, andblock the interaction of PVRIG with its binding partner, PVRL2. This wasachieved by panning a human fab fragment phage display library against arecombinant protein comprising the human PVRIG extracellular domain(ECD) fused to the human IgG1 Fc region, and screening the resultingantibodies for their ability to block the PVRIG interaction with PVRL2.

Protocols

Functional QC of reagents: The purity of the panning reagent, PVRIG ECDfused to human IgG1 Fc domain (PVRIGHH), was determined by MicrofluidicsCapillary Electrophoresis using a LabChip System (PerkinElmer). Activityof the panning reagent was validated by its ability to bind its ligandPVRL2.

ELISA to detect protein-protein interaction: His-tagged PVRL2recombinant protein was diluted to 2 μg/mL in phosphate buffered saline(PBS) and 50 μL aliquots were coated on the wells of a high bindingEIA/RIA plate (Costar) overnight at 4° C. Coated plate wells were rinsedtwice with PBS and incubated with 300 μL blocking buffer (5% skim milkpowder in PBS pH 7.4) at room temperature (RT) for 1 hr. Blocking bufferwas removed and plates were rinsed twice more with PBS. Plate-boundPVRL2 was incubated with varying concentrations of PVRIGHH in solution(linear range of 0.1 μg/mL to 4 μg/mL in a 50 μL/well volume) at RT for1 hr. Plates were washed three times with PBS-T (PBS 7.4, 0.05%Tween20), then three times with PBS and 50 μL/well of a HRP-conjugatedsecondary antibody was added (Human IgG Fc domain specific). This wasincubated at RT for 1 hr and plates were washed again. ELISA signalswere developed in all wells by adding 50 μL of Sureblue TMB substrate(KPL Inc) and incubating for 5-20 mins. The HRP reaction was stopped byadding 50 μL 2N H2SO4 (VWR) and absorbance signals at 450 nm were readon a SpectraMax (Molecular Devices) or EnVision (PerkinElmer)spectrophotometer.

Preparation of biotinylated PVRIG: To facilitate phage panning insolution using streptavidin-coated magnetic beads, PVRIG H:H and anirrelevant human IgG1 Fc isotype control were biotinylated usingLightning-Link® Biotin kit (Innova Biosciences). Biotinylation reactionswere performed following the manufacturer's protocol and thebiotinylated reagents were stored at 4° C. for further QC andbiopanning. The purity and activity of the biotin-labeled proteins wasassessed by LabChip and functional ELISA, as described in Section 2.1.In addition, the degree of biotinylation was assessed by ELISA using twoapproaches: 1) the biotinylated reagents were adsorbed on a high bindingEIA/RIA plate and the proteins were detected using HRP-conjugatedstreptavidin, and 2) the biotinylated proteins were incubated on EIA/RIAplate pre-coated with streptavidin and the binding was detected using aHRP-conjugated human IgG Fc domain specific secondary antibody.

Phage panning of human antibody library: Panning reactions were carriedout in solution using streptavidin-coated magnetic beads to capture thebiotinylated antigens. Note that all washing and elution steps wereconducted using a magnetic rack to capture the beads (Promega). Allincubation steps were conducted at room temperature with gentle mixingon a tube rotator (BioExpress). Four panning sub-campaigns wereconducted, each with a different combination of antigen concentrations,washes and Fc-binder depletion steps (Table 1).

All the panning sub-campaigns were carried out using the biotinylatedPVRIG H:H antigen. For each round of panning, the phage libraries weredepleted against 100 pmol of an irrelevant human IgG1 Fc protein in twosuccessive steps. Following depletion, sub-campaigns A and B involvedpanning against 50 nM of the antigen in each round, under low and highstringency wash conditions, respectively. Sub-campaigns C and D wereidentical to sub-campaign B, except that in campaign C the library wasblocked with 10-fold excess of the irrelevant IgG1 Fc protein in panningrounds 2 and 3. Sub-campaign D differed in that 5 nM antigen was used inround 3.

TABLE 1 Antigen and washing conditions used for phage panning againstPVRIG H:H. Sub- Antigen campaign Concen- Round tration Washes FcDepletion A 1 50 nM 3x PBS-T + 3x PBS 2X 100 pmol 2 50 nM 3x PBS-T + 3xPBS 2X 100 pmol 3 50 nM 3x PBS-T + 3x PBS 2X 100 pmol B 1 50 nM 3xPBS-T + 3x PBS 2X 100 pmol 2 50 nM 6x PBS-T + 6x PBS 2X 100 pmol 3 50 nM6x PBS-T + 6x PBS 2X 100 pmol C 1 50 nM 3x PBS-T + 3x PBS 2X 100 pmol 250 nM 6x PBS-T + 6x PBS 2X 100 pmol + block with 1 nmol 3 50 nM 6xPBS-T + 6x PBS 2X 100 pmol block with 1 nmol D 1 50 nM 3x PBS-T + 3x PBS2X 100 pmol 2 50 nM 6x PBS-T + 6x PBS 2X 100 pmol 3  5 nM 6x PBS-T + 6xPBS 2X 100 pmol

2.4. Preparation of phage library for panning: All phage panningexperiments used the XOMA031 human fab antibody phage display library(XOMA Corporation, Berkeley, Calif.). Sufficient phage for a 50-foldover-representation of the library were blocked by mixing 1:1 with 10%skim milk powder in PBS (final skim milk concentration 5%) andincubating for 1 hr.

2.4.1. Antigen coupling to streptavidin beads: For each sub-campaign,three 100 μL aliquots of Dynal streptavidin-coated magnetic beads (LifeTechnologies) were blocked by suspension in 1 mL of blocking buffer (5%skim milk powder in PBS) and incubated for 30 inns. One blocked beadaliquot was mixed with 100 pmols of biotinylated PVRIG H:H. The othertwo aliquots were mixed with 100 pmols of the irrelevant antigen fordepletion of Fc-only binders. Biotin-labeled antigens were coupled tothe beads for 30 mins at RT. Bead suspensions were washed twice with PBSto remove free antigen and re-suspended in 100 μL blocking buffer.

2.4.2. Depletion of human IgG1 Fe and streptavidin bead binders from thephage library: It was necessary to remove unwanted binders tostreptavidin beads and the Fc region of PVRIGHH before phage panningcould commence. To achieve this, blocked phage was mixed with one 100 μLaliquot of uncoupled streptavidin beads and incubated for 45 mins. Thebeads (and presumably unwanted bead and human IgG1 Fc-binders) werediscarded. This step was repeated once and depleted phage librarysupernatants were reserved for panning.

2.5. Phage panning round 1: The blocked and depleted phage library wasmixed with biotinylated PVRIG H:H coupled to magnetic beads describedabove. This suspension was incubated for 1 hr at RT with gentle rotationto allow binding of PVRIG H:H specific phage. Non-specific binders wereremoved by washing according to the protocol in Table 1. After washing,bound phage were eluted by incubation with 500 μL of 100 mMtriethylamine (TEA) (EMD) for 15 mins at RT. The eluate was neutralizedby adding 500 μL of 1 M Tris-HCl pH 8.0 (Teknova).

2.5.1. Determination of phage titer: 10 μL of the initial phage library(input titer) or panning eluate (output titer) was serially diluted(10-fold) in PBS. A 90 μL aliquot of each phage dilution was mixed with500 μL of TG1 E. coli cells grown to an optical density of ˜0.5 at 600nm (OD 600 nm). Phage were allowed to infect the cells by stationaryincubation for 30 mins, then shaking incubation (250 rpm) for 30 mins,all at 37° C. A 10 μL aliquot of each infected cell culture was spottedon a 2YT agar plate supplemented with 2% glucose and 100 μg/mLcarbenicillin (2YTCG, Teknova). Plates were incubated overnight at 30°C. Colonies growing from each 10 μL spot were counted and used tocalculate input and output titers.

2.5.2. Phage rescue: The remaining phage eluate (˜1 mL) was mixed with10 mL of TG1 E. coli cells grown to an OD 600 nm of 0.5. Phage wereinfected into cells as detailed in section 2.5.1. Infected cells werepelleted by centrifugation at 2500×G, re-suspended in 750 μL 2YT medium(Teknova) and spread on 2YTCG agar plates. These were incubatedovernight at 37° C. and the resulting E. coli lawns were scraped andre-suspended in ˜20 mL liquid 2YTCG (Teknova). A small aliquot ofre-suspended cells was inoculated into 50 mL 2YTCG to achieve an OD 600nm of 0.05, and then grown at 37° C. with 250 rpm shaking until the ODreached 0.5. The resulting culture was infected with M13K07 helper phage(New England Biolabs) and incubated overnight at 25° C. with shaking toallow phage packaging. The culture supernatant containing rescued phageparticles was cleared by centrifugation at 2500×G and 1 mL was carriedover for either a) a subsequent round of panning or b) fab bindingscreens.

Phage panning rounds 2-3: Second and third rounds of panning wereconducted as per the steps above, except that the rescued phagesupernatant from the previous round was used in place of the phagelibrary. The washing conditions, depletion and the antigenconcentrations used are listed in Table 1.

2.6. Binding Screens Using Fabs Prepared from Periplasmic Extracts

2.6.1. Fab expression vectors: The XOMA031 library is based on phagemidconstructs that also function as fab expression vectors. These vectorscontain fab heavy chain and light chain expression cassettes, a lacpromoter to drive expression of the antibody genes, and an ampicillinresistance gene. The antibody chains are appended with N-terminal signalpeptides to drive their secretion into the periplasmic space. TheC-terminal of the heavy chain carries a truncated gene III proteinsequence for incorporation into phage particles. The heavy chain alsocarries hexa-histidine (SEQ ID NO: 7), c-myc and V5 affinity tags.Transformation of these vectors into E. coli and induction withisopropyl R-D-1-thiogalactopyranoside (IPTG) results in periplasmicexpression of soluble fab molecules.

2.6.2. Fab PPE production: Eluted phage pools from panning round 3 werediluted and infected into TG1 E. coli cells (Lucigen) so that singlecolonies were generated when spread on a 2YTCG agar plate. This resultedin each colony carrying single fab clone. Individual clones wereinoculated into 1 mL 2YTCG starter cultures in 96-well deep well blocks(VWR) using a Qpix2 instrument (Molecular Devices). These startercultures were grown overnight in a Multitron 3 mm incubator (Infors) at37° C. with 700 rpm shaking. For fab expression, 20 μL of 1 mL startercultures were transferred into a second set of deep well platescontaining 1 mL 2YT with 0.1% glucose and 100 μg/mL ampicillin. Cultureswere grown until the average OD 600 nm was 0.5-1.0 and proteinexpression was induced by adding IPTG (Teknova) to a final concentrationof 1 mM. Expression cultures were incubated overnight in the Multitroninstrument at 25° C. with 700 rpm shaking.

Fab proteins secreted into the E. coli periplasm were extracted foranalysis. Cells were harvested by centrifugation at 2500×G, thesupernatants were discarded and pellets were re-suspended in 75 μLice-cold PPB buffer (Teknova). Extracts were incubated for 10 inns at 4°C. with 1000 rpm shaking, and 225 μL ice-cold ddH2O was added andincubated for a further hr. The resulting periplasmic extract (PPE) wascleared by centrifugation at 2500×G and transferred to separate platesor tubes for ELISA and FACS analysis. All extraction buffers containedEDTA-free Complete Protease Inhibitors (Roche).

Each plate of samples also included duplicate “blank PPE” wells to serveas negative controls. These were created by intentionally leaving two 1mL cultures un-inoculated and then processing them in the same way asthe fab PPEs, thereby creating a sample with no bacterial growth andtherefore no fab expression.

2.6.3. Primary screen by ELISA: Two 96-well plates of PPE extracts persub-campaign were tested for binding to PVRIG H:H by ELISA. Note that anon-biotinylated version of the protein was used for the ELISA screen toavoid the selection of biotin or streptavidin-binders. PVRIGHHrecombinant protein was diluted to 2 μg/mL in phosphate buffered saline(PBS) and 50 μL aliquots were coated on the wells of a high bindingEIA/RIA plate (Costar) overnight at 4° C. Coated plate wells were rinsedtwice with PBS and incubated with 300 μL blocking buffer (5% skim milkpowder in PBS pH 7.4) at room temperature (RT) for 1 hr. Blocking bufferwas removed and plates were rinsed twice more with PBS. Plate-boundPVRIG was incubated with the PPEs, pre-blocked with 3% skim milk, at RTfor 1 hr. Plates were washed three times with PBS-T (PBS 7.4, 0.05%Tween20), then three times with PBS and 50 μL/well HRP-conjugated,anti-human Fab secondary antibody (Jackson ImmunoResearch) was added ata 1:2000 dilution in 5% milk in PBS. This was incubated at RT for 1 hrand plates were washed again. ELISA signals were developed in all wellsby adding 50 μL of Sureblue TMB substrate (KPL Inc) and incubating for5-20 mins. The HRP reaction was stopped by adding 50 μL 2N H2SO4 (VWR)and absorbance signals at 450 nm were read on a SpectraMax (MolecularDevices) or EnVision (PerkinElmer) spectrophotometer. Wells that showedsignal over background (blank PPE) ratio >3 were selected as positivehits.

2.6.4. Sequence analysis of ELISA positive fabs: The positive hits fromthe ELISA screen were selected and re-arrayed into a new 96-well plate.The clones were grown overnight at 37° C. and the plasmid DNA wassequenced using heavy chain and light chain-specific primers. Thesequences were assembled and analyzed using Xabtracker (XOMA) software.The clones were deemed sequence-unique if there were more than onenon-conservative differences in the heavy chain CDR3. Clones with sameor similar heavy chain but significantly different light chains werelabeled as siblings of the original clone.

2.6.5. FACS screening of fabs as PPEs: The sequence-uniqueELISA-positive fab clones were selected and analyzed for their abilityto bind PVRIG over-expressing cells by fluorescence-activated cellsorting (FACS). Analyses were conducted using HEK293 cellsover-expressing the human PVRIG antigen. In a parallel experiment,un-transfected HEK293 cells were used as a negative control for each fabsample.

The PPEs for the sequence-unique ELISA-positive fab clones weregenerated as described above. All the assays were conducted using FACSbuffer (1% BSA and 0.1% sodium azide in PBS). The human PVRIG andun-transfected HEK293 cells were harvested, washed twice andre-suspended at a density of 2×10⁶ cells/ml. A 25 μl aliquot of cellswas mixed with 25 μl of each PPE sample and incubated for 1 hr at 4° C.with gentle shaking. Two blank PPE controls were also included in theanalysis. Plates were washed one time in 200 μl of FACS buffer and 50 μLof a 2 μg/mL dilution of a mouse anti-C-myc antibody (Roche) was addedto each well. After incubation for 30 mins at 4° C., cells were washedagain and 25 μl of a 5 μg/mL dilution of goat anti mouse fab-AF647(Jackson Immunoresearch) was added to each PPE and negative controlwell. All secondary antibodies were incubated for 30 min at 4° C. Aftertwo washes, cells were re-suspended in a final volume of 50 μl offixation buffer (2% paraformaldehyde in FACS buffer). Samples were readon an Intellicyt HTFC screening system, recording approximately 5000events per well in a designated live gate. Data was analyzed usingFlowJo (De Novo Software, CA, USA) and exported to Excel. Ratio of MeanFluorescence Intensity (MFI) for the human PVRIG over-expressing HEKcells and the un-transfected 293 cells was calculated using Xabtrackersoftware (XOMA). Positive hits on each plate were identified as thosegiving an MFI ratio 5-fold greater than the averaged blank PPE controlsignal.

Re-formatting of fab hits and production as human IgG molecules:Potential PVRIG binding fabs were converted to full length human IgGsfor further characterization. Protein expression constructs were derivedby PCR-amplification of variable heavy, lambda and kappa domain genes,which were sub-cloned into pFUSE-CHIg-hG1 (human IgG1 heavy chain),pFUSE2-CLIg-hK (human kappa light chain) or pFUSE2-CLIg-hL2 (humanlambda 2 light chain) vectors, respectively (all expression vectors weresourced from Invivogen).

Expi293 cells (Life Technologies) were seeded at 6×10⁵ cells/ml inExpi293 medium (Life Technologies) and incubated for 72 hrs at 37° C. ina humidified atmosphere of 8% CO2 with shaking at 125 rpm. This cellstock was used to seed expression cultures at 2.0×10⁶ cells/ml inExpi293 medium. These cultures were incubated as above for 24 hrs withshaking at 135 rpm.

For transfection, cells were diluted again to 2.5×10⁶ cells/ml inExpi293 medium. The protein expression constructs for antibody heavychain and light chain were mixed at a ratio of 1:2. For every 30 mL ofexpression culture volume, 30 pg of DNA and 81 μL of Expifectamine (LifeTechnologies) were each diluted separately to 1.5 mL with Opti-MEM (LifeTechnologies) and incubated for five minutes. Diluted DNA andExpifectamine were then mixed and incubated at RT for 20 mins. This wasthen added to the expression culture in a shaker flask and incubated asdescribed above, with shaking at 125 rpm.

Approximately 20 hrs post-transfection, 150 μL of ExpiFectamine 293transfection Enhancer 1 and 1.5 mL of ExpiFectamine 293 TransfectionEnhancer 2 was added to each flask. Cultures were incubated for afurther five days (six days post-transfection in total) and supernatantswere harvested by centrifugation. IgGs were purified from thesupernatants using an AKTA Pure FPLC (GE Healthcare Bio-Sciences) andHiTrap MabSelect Sure affinity columns (GE Healthcare Bio-Sciences)according to manufacturer's instructions.

FACS screening of reformatted IgG1 antibodies: FACS screening of thereformatted antibodies was done similarly to the PPE based screendescribed herein, except that a dose-dependent titration of the purifiedantibodies was performed. The human PVRIG over-expressing HEK293 cells,or the un-transfected HEK293 cells, were incubated with varyingconcentrations (0-10 μg/ml) of the anti PVRIG antibodies or isotypecontrols in FACS buffer at 4° C. for 60 mins. Cells were washed once inFACS buffer, re-suspended in 50 μl of Alexa Fluor 647 conjugatedanti-human IgG (Fab fragment specific) diluted 1:200 and incubated for30 mins at 4° C. in the dark. Cells were washed twice and re-suspendedin a final volume of 80 μl of FACS buffer and Propidium Iodide(Biolegend cat #421301) diluted 1:1000. Samples were analyzed using anIntellicyt HTFC screening system (Intellicyt). Data was analyzed usingFlowJo (DeNovo), exported to Excel (Microsoft) and plotted in GraphPadPrism (GraphPad Software, Inc.).

Results

Functional QC of the PVRIG H:H recombinant protein: The purity of thePVRIG H:H protein was assessed by microfluidics capillaryelectrophoresis using a LabChip system. Under reducing conditions, therecombinant protein migrated at 80 kDa, consistent with its calculatedmolecular weight of 80.4 kDa, and showed 99% purity (data not shown).Under non-reducing conditions, one additional peak was observed whichlikely resulted from the presence of a dimeric form of the protein dueto Fc-Fc interaction.

The functional integrity of the recombinant protein was assessed byevaluating its binding to PVRL2 (a known ligand for PVRIG) in ELISA. Adose-dependent response was observed for the binding of PVRIG H:H toPVRL2 (data not shown). In comparison, no binding was observed for airrelevant human IgG1 Fc control. Taken together, this indicated thatthe PVRIG H:H recombinant protein is of high purity and is functionallyactive, and thus is suitable for biopanning.

QC of the biotinylated PVRIG H:H recombinant protein: The purity of thebiotinylated PVRIGHH protein was assessed by microfluidics capillaryelectrophoresis using LabChip system. No significant differences wereobserved between the non-biotinylated and the biotinylated recombinantproteins (data not shown). Note that an additional 44.3 kDa peakobserved in the biotinylated protein sample. This peak may result fromthe monomeric form of the PVRIG H:H protein or maybe an artifact of thequenching reaction of the biotinylation kit.

Successful biotinylation was confirmed by incubating the biotinylatedprotein on a streptavidin-coated EIA plate and detecting the boundprotein using a HRP-conjugated anti human IgG1 Fc secondary antibody.The binding of biotinylated PVRIG H:H to the streptavidin-coated EIAplate was comparable to a commercially sourced irrelevant biotinylatedprotein (data not shown).

Phage panning: The biotinylated PVRIG H:H protein was used for phagepanning against the XOMA031 human fab antibody phage display library(XOMA Corporation, Berkeley, Calif.). Three rounds of biopannings wereperformed, under 4 different combinations of washing stringency, antigenconcentration, and depletion of Fc binders (sub-campaigns A-D). Thesuccess of each round was estimated using the phage output titers.Qualitative guidelines were used to define the success of the panningsub-campaigns, such as significant reduction in phage titers after round1, increase or maintenance of phage titers after rounds 2 and 3, anddecrease in phage titers upon increasing wash stringency or decreasingantigen concentration. All 4 sub-campaigns resulted in phage titers inthe expected range that were consistent among the sub-campaigns (datanot shown).

Screening of phage output as fab PPEs: Two 96-well plates of fab clones(as PPEs) for each of the four sub-campaigns were screened to evaluatethe success of biopanning. The results are summarized in table 3 and arediscussed in further detail below. Overall, all 4 sub-campaigns yieldedsignificant numbers of PVRIG H:H specific fabs. A total of 49target-specific unique fabs were identified. The sub-campaigns B and Dshowed the highest ELISA hit rates and FACS correlation and wereselected for an extended screen.

TABLE 3 Summary of pilot screen of fab PPEs. For each sub-campaign, thetotal number of clones screened, ELISA hits, FACS hits and sequenceuniqueness are listed. Open reading frames (ORFs) represent the clonesthat were successfully sequenced as a full-length fab. Specificity isbased on the lack of non-specific binding to irrelevant proteins inELISA. FACS correlation represents the percent of ELISA hits that werealso FACS positive (specifically bound to PVRIG over-expressing HEK293cells). Sub A Sub B Sub C Sub D Overall Clones screened 182 182 182 182728 ELISA positive (>3 S/N) 48 51 44 68 211 ELISA Hit rate 26% 28% 24%37% 29% ORFs 36 (75%) 45 (88%) 35 (80%) 63 (93%) 179 (85%) Uniquesequences 25 21 17 31 73 Diversity 69% 47% 49% 49% 41% Specificity byELISA* 100%  100%  100%  100%  100%  FACS Binders (>5 S/N) 14 17 14 2449** FACS correlation 56% 81% 82% 77% 67% *No non-specific binding toirrelevant Fc conjugates or PVRL2; **35 unique HCs, 14 siblings

Primary fab screen (ELISA): Two 96-well plates (182 fab clones) of PPEsfor each sub-campaign were screened by ELISA against the PVRIG H:Hrecombinant protein. Note that although biotinylated protein was usedfor panning, the non-biotinylated version was used for the ELISA screen,which avoided detection of biotin or streptavidin-specific binders. The4 sub-campaigns resulted in ELISA hit rates ranging from 24-37% when thethreshold for a ‘positive’ signal was set at a 3-fold ratio oftarget-specific binding: blank PPE control signal.

Secondary screen (DNA sequence analysis, ELISA and FACS) fabs: The ELISApositive clones were sequenced to select non-redundant fabs.Seventy-three sequence-unique fab clones were identified. 19 clones wereunique to sub-campaign A, 13 clones were unique to sub-campaign B, 10clones were unique to sub-campaign C, 18 clones were unique tosub-campaign D, while the remaining 23 clones were shared between thecampaigns. Sequence-unique, ELISA-positive fab clones were re-expressedas PPEs and screened for specific binding by FACS. A total of 49 out of73 unique clones were identified as PVRIG specific ELISA and FACSbinders (following the criteria established in 2.6.5). The 49 FACSbinders corresponded to 35 antibodies with unique heavy chains and 14siblings that have unique light chains but share the heavy chain withone of the unique clones. A summary of FACS binding data is presented inTable 4.

The sequence unique fabs were also tested for non-specific binding. Allthe fab PPEs analyzed bound to the PVRIG H:H recombinant protein with anassay signal greater than 3-fold over the blank PPE control. In aparallel assay, fab PPEs were tested for binding to two irrelevantproteins with the same IgG1 Fc region, as well as the PVRL2 recombinantprotein. None of the clones showed significant non-specific binding tothe controls, suggesting that the selected fabs are specific for PVRIG.

TABLE 4 FACS binding summary for PVRIG fabs. All unique ELISA positivefabs were analyzed by FACS. The mean fluorescence intensity (MFI) wasmeasured for the PVRIG over-expressing HEK293 cells as well as theun-transfected HEK293 cells. The MFI ratio for the target- specific vsoff-target binding was calculated. Clones with MFI ratio >5 wereselected as hits and are listed below. fab clone MFI ratio CPA.7.001 11CPA.7.002 8.9 CPA.7.003 9.5 CPA.7.004 9.3 CPA.7.005 6.5 CPA.7.006 9.6CPA.7.007 14 CPA.7.008 14 CPA.7.009 10 CPA.7.010 7.6 CPA.7.011 10CPA.7.012 19 CPA.7.013 12 CPA.7.014 14 CPA.7.015 15 CPA.7.016 7.6CPA.7.017 13 CPA.7.018 7.8 CPA.7.019 16 CPA.7.020 6.9 CPA.7.021 15CPA.7.022 7.5 CPA.7.023 12 CPA.7.024 9.8 CPA.7.025 6 CPA.7.026 5.3CPA.7.027 9.2 CPA.7.028 17 CPA.7.029 6.7 CPA.7.030 15 CPA.7.031 8.5CPA.7.032 7.6 CPA.7.033 22 CPA.7.034 7.7 CPA.7.035 14 CPA.7.036 5CPA.7.037 5.3 CPA.7.038 6.3 CPA.7.039 12 CPA.7.040 12 CPA.7.041 7.6CPA.7.042 5.4 CPA.7.043 13 CPA.7.044 7.9 CPA.7.045 7.8 CPA.7.046 10CPA.7.047 8.4 CPA.7.049 10 CPA.7.050 22

Reformatting of the ELISA and FACS positive fabs into hIgG1:All uniqueELISA and FACS binders were reformatted for expression as human IgG1molecules in Expi293 cells. Out of the original 49 antibodies, 44 weresuccessfully expressed as full-length antibodies. These reformattedantibodies were tested for retained binding to PVRIG over-expressingHEK293 cells alongside an irrelevant human IgG1 isotype control. Allantibodies were also tested against un-transfected HEK293 cells. Theresulting binding results were used to demonstrate the specificity ofthe antibodies and also plotted to calculate the equilibrium bindingconstant (KD). Nine out of the remaining 44 antibodies showed weakbinding or significant non-specific binding. The remaining 35 antibodieswere selected for further analysis in cell-based functional assays. TheFACS-based KD of these antibodies are listed in Table 6. The KD valuesrange from 0.30 nM to 96 nM, with a median of 9.4 nM, suggesting thatmost antibodies obtained from the panning campaign are very specific andbind to PVRIG with high affinity.

TABLE 5 Expression and binding summary of reformatted antibodies. Allunique ELISA and FACS positive fabs were reformatted into the human IgG1backbone. FACS KD values were determined by dose titration against thePVRIG over-expressing HEK293 cells. Off-target binding was determined bydose titration against the un-transfected HEK293 cells. Antibody FACS KD(nM) CPA.7.001 No-expression CPA.7.002 44.35 CPA.7.003 Non-specificbinding CPA.7.004 21.71 CPA.7.005 95.56 CPA.7.006 No-expressionCPA.7.007 0.73 CPA.7.008 No-expression CPA.7.009 33.00 CPA.7.010 21.89CPA.7.011 66.02 CPA.7.012 0.30 CPA.7.013 No-expression CPA.7.014 2.04CPA.7.015 1.34 CPA.7.016 22.02 CPA.7.017 1.82 CPA.7.018 9.29 CPA.7.0190.45 CPA.7.020 86.97 CPA.7.021 11.22 CPA.7.022 4.17 CPA.7.023 4.08CPA.7.024 9.08 CPA.7.025 Non-binder CPA.7.026 Non-binder CPA.7.027Non-binder CPA.7.028 7.14 CPA.7.029 Weak binding CPA.7.030 No-expressionCPA.7.031 Non-binder CPA.7.032 8.78 CPA.7.033 12.8 CPA.7.034 14.2CPA.7.035 Non-binder CPA.7.036 6.0 CPA.7.037 Non-specific bindingCPA.7.038 20.26 CPA.7.039 3.76 CPA.7.040 0.79 CPA.7.041 52.2 CPA.7.04224.26 CPA.7.043 13.2 CPA.7.044 9.4 CPA.7.045 3.73 CPA.7.046 Non-specificbinding CPA.7.047 5.36 CPA.7.049 19.9 CPA.7.050 68.3

Summary and Conclusions

A phage display antibody discovery campaign was conducted to isolatebinders against the immuno-oncology target PVRIG using a recombinantFc-tagged version of the antigen. Quality control analysis showed thatthe panning antigen was pure and functionally active. The panning effortyielded 49 unique fab clones that specifically bound to the PVRIGtarget, both as a recombinant protein and on the cell surface. Of these,35 were successfully produced as human IgG1 antibodies and were shown toretain specific binding to the PVRIG. This pool of antibodies displayedhigh affinities in a FACS assays, with 18 out of 35 antibodies bindingwith a KD<10 nM.

Example 10 Demonstration of the Ability of the Anti-Human PVRIG Fabs toBlock the Interaction Between PVRIG and PVRL2 by ELISA

Method: The human PVRL2-His (Catalog #2229-N2-050/CF, R&D Systems), wascoated on the ELISA plate. Fab periplasmic extracts (PPEs), diluted 1:1in 5% skim milk, were preincubated with 1 μg/ml (final concentration) ofthe human PVRIG-Fc, for 15 min at RT. The fab-receptor mixture wasallowed to bind the PVRL2-His coated on the ELISA plate. ThePVRIG-Fc/PVRL2-His interaction was probed using anti-human Fc antibody,conjugated to HRP (Jackson Immuno Research catalog #709-035-098). In theabsence of PPE (negative wells), a strong positive signal was expected.For blocking fabs, the signal would be significantly reduced. The fabclones with >5-fold lower signal than the negative wells (>80% blocking)could be selected as blocking fabs.

Protocol:

ELISA plates (Costar 9018) were coated with 50 ul of 2 g/ml antigen andwere stored at 4 C overnight. The antigen-coated plates were washed 3times with 1×PBS. The plate was blocked with 200 μl of 5% skim milk inPBS and incubated 1 hr at RT (room temperature). Next the plate waswashed with 1×PB.

After adding 50 μl/well of Fab PPEs (diluted in 5% skim milk), the platewas preincubated with 1 μg/ml of the human PVRIG-Fc that was added tothe respective wells. The “no fab” control was performed with 2 wells.

The plate was incubated 1 hr at RT.

The plates were washed 3 times with 1×PBST and 3 times with 1×PBS.

After adding 50 μl/well of the HRP-conjugated secondary antibody(Jackson Immuno Research, 709-035-098), diluted in 5% milk in PBS, theplate was incubated 1 hr at RT.

The plates were washed 3 times with 1×PBST and 3 times with 1×PBS.

After adding 50 μl/well of the TMB substrate and waiting until the colordevelops, the reaction was stopped by adding 50 μl/well of 2N H2SO4.Absorbance was measured at 450 nm.

Results

FIGS. 52A-52B shows the results of testing anti-PVRIG antibodies fortheir ability to block at least 80% of PVRL2 binding to PVRIG. As shown,a large number of such antibodies were able to successfully block atleast 80% of the binding. Specifically the antibodies which blockedsuccessfully are designated as follows:

CPA.7.001, CPA.7.003, CPA.7.004, CPA.7.006, CPA.7.008, CPA.7.009,CPA.7.010, CPA.7.011, CPA.7.012, CPA.7.013, CPA.7.014, CPA.7.015,CPA.7.017, CPA.7.018, CPA.7.019, CPA.7.021, CPA.7.022, CPA.7.023,CPA.7.024, CPA.7.033, CPA.7.034, CPA.7.036, CPA.7.040, CPA.7.046,CPA.7.047, CPA.7.049, CPA.7.050,

Example 11: Surface Plasmon Resonance Study of Epitope Binning of 37Anti PVRIG IgG Antibodies Binding to Human PVRIG Fusion Protein

Materials and Methods

Experiments were performed using a ProteOn XPR 36 instrument at 22° C.with all samples kept at 4° C. during the experiment.

Step 1: The following anti-PVRIG mAbs were each diluted to ˜10 μg/mL in10 mM sodium acetate, pH 5.0 and covalently immobilized on independentspots on a ProteOn GLC biosensor chip using standard amine coupling:

CPA.7.002 CPA.7.017 CPA.7.033 CPA.7.003 CPA.7.018 CPA.7.034 CPA.7.004CPA.7.019 CPA.7.036 CPA.7.005 CPA.7.020 CPA.7.037 CPA.7.007 CPA.7.021CPA.7.038 CPA.7.009 CPA.7.022 CPA.7.039 CPA.7.010 CPA.7.023 CPA.7.040CPA.7.011 CPA.7.024 CPA.7.043 CPA.7.012 CPA.7.026 CPA.7.045 CPA.7.014CPA.7.028 CPA.7.046 CPA.7.015 CPA.7.029 CPA.7.047 CPA.7.016 CPA.7.032CPA.7.050

The activation step occurred in the horizontal flow direction for fiveminutes while the immobilization step occurred in the vertical flowdirection. MAbs were injected for four minutes after surface activation.The blocking step occurred in both the vertical and horizontal positionsat five minutes each so that the horizontal “interspots” could be usedas reference surfaces. MAbs were immobilized at a range of ˜450 RU-5000RU. An additional mAb CPA.7.041 was also binned in this study, but onlyas an analyte in solution. See below.

Step 2: Preliminary experiments involved several cycles of injecting ˜20nM PVRIG antigen (PVRIG H:H-2-1-1 #448, GenScript) over all immobilizedmAbs for three minutes at a flow rate of 25 μL/min followed byregeneration with a 30-second pulse of 10 mM glycine-HCl, at either pH2.0 or pH 2.5, depending on the horizontal row of mAbs in the GLC chiparray. Antigen samples were prepared in degassed PBST (PBS with 0.05%Tween 20) running buffer with 100 μg/mL BSA. These preliminaryexperiments showed that clones CPA.7.026 and CPA.7.029 did not bind tothe antigen and were therefore not binned. The remaining mAbs on theProteOn array showed reproducible binding to the antigen.

Step 3: A “pre-mix” epitope binning protocol was performed because ofthe bivalency of the fc-fusion PVRIG antigen. In this protocol each mAblisted in Step 1, plus mAb CPA.7.041, was pre-mixed with PVRIG antigenand then injected for three minutes over all immobilized mAbs. The molarbinding site concentration of each mAb was in excess of the molarantigen binding site concentration. The final binding site concentrationof each mAb was ˜400 nM and the final binding site concentration of theantigen was −20 nM. An antigen-only control cycle was performed aftervery eight mAb injection cycles to monitor the activity of theimmobilized mAbs throughout the experiment. Buffer blank injections werealso performed after about every eight mAb injection cycles fordouble-referencing. Additional controls included each mAb injected aloneover all immobilized mAbs at concentrations identical to the pre-mixinjection cycles. All surfaces were regenerated with a 30 second pulseof 10 mM glycine-HCl at either pH 2.0 or pH 2.5 depending on which rowof mAbs in the array was being regenerated, and all cycles were run at aflow rate of 25 μL/min. MAb and antigen samples were prepared indegassed PBST running buffer with 100 μg/mL BSA.

Step 4: Sensorgram data were processed and referenced using ProteOnManager Version 3.1.0.6 using interspots and buffer blanks fordouble-referencing. The mAb-only control injections were used as theinjection references where significant binding with the mAb-onlyinjections was observed. An antibody pair was classified as having ashared antigen binding epitope (designated as a red “0” in the matrix inFIG. 43) if no binding was observed from the injection of mixed mAb andantigen over the immobilized mAb, or if binding was significantlyreduced as compared to the antigen-only control injection over the sameimmobilized mAb. An antibody pair was classified as binding to differentantigen epitopes, or “sandwiching” the antigen (designated as a green“1” in the matrix in FIG. 43) if the injection of mixed mAb and antigenshowed binding to the immobilized mAb similar to or greater than theantigen-only control over the same immobilized mAb.

Step 5: The blocking pattern for mAb CPA.7.041 (#37) was studied only asan analyte because the GLC chip array has only 36 spots. Therefore forconsistency, hierarchical clustering of the binding patterns in thebinary matrix for each mAb pre-mixed with antigen (vertical patterns inFIG. 42) was performed using JMP software version 11.0.0. The blockingpatterns of the immobilized mAbs (horizontal patterns in FIG. 42) werealso clustered as a comparison to the blocking patterns of the mAbspre-mixed in solution (data not shown, see Results for discussion).

Results: FIG. 42 shows the binary matrix of the blocking (“0”) orsandwiching (“1”) between each mAb pair where the mAbs are listed inidentical order both vertically (mAbs on the surface—“ligands”) andhorizontally (mAbs in solution—“analytes”). Identical “bins” of blockingpatterns for all mAbs as analytes are highlighted in FIG. 42 with ablack box around each group of similar vertical patterns. FIG. 43 showsthe dendrogram of the vertical (analyte) blocking patterns in the matrixin FIG. 42. For the strictest definition of an epitope “bin” where onlythose mAbs which show identical blocking patterns technically bintogether, there are a total of 4 discrete bins. Specifically, 33 of the35 mAbs that were binned comprise two bins where the only differencebetween these two bins is whether a mAb sandwiches (Bin 2, see FIG. 42and FIG. 43) with or blocks (Bin 1, see FIG. 42 and FIG. 43) binding toCPA.7.039. This means that CPA.7.039 is in its own separate bin. Thefourth bin consists only of mAb CPA.7.050 which is unable to blockantigen binding to any of the other 34 mAbs. Hierarchical clustering ofthe blocking patterns of the mAbs as ligands (horizontal patterns inFIG. 42) showed mAb CPA.7.016 sandwiching antigen with mAb CPA.7.039whereas as an analyte it blocks antigen binding to immobilizedCPA.7.039. Hence clone CPA.7.016 would be placed in bin 2 rather than inbin 1. The mAbs in each bind are listed in FIG. 43. Processed sensorgramdata representative of each bin are shown in FIG. 44 to FIGS. 47A-47JJ.

Summary: 35 anti-PVRIG IgG mAbs were binned using SPR according to theirpair-wise blocking patterns with fc fusion human PVRIG. By the strictestdefinition of an epitope bin, there area total of four discrete bins. 33of the 35 mAbs comprise two bins which differ only by whether theirrespective component mAbs block or sandwich antigen with cloneCPA.7.039.

Example 12 Surface Plasmon Resonance Kinetic Screen of 50 Anti-PVRIGHuman Fabs Prepared in Periplasmic Extracts

Materials and Methods

All experiments were performed using a Biacore 3000 instrument and aProteOn XPR 36 instrument at 22° C.

Step 1: The molar concentration of all 52 fabs in periplasmic extractsupernatant were quantitated using a Biacore 3000 instrument at 22° C.Each fab was diluted 20-fold and then injected for 2 minutes at 5 μL/minover high density anti-human fab (GE Healthcare 28-9583-25) surfacesprepared using standard amine coupling with a CM5 Biacore chip (GEHealthcare). A standard human fab at a known concentration (BethylP80-115) was then injected over the anti-fab surface with the sameconditions as the fab supernatants. Samples were prepared in the runningbuffer which was degassed HBSP (0.01 M HEPES, 0.15 M NaCl, 0.005% P20,pH 7.4) with 0.01% BSA added. The association slopes of each SPRsensorgram from each fab supernatant was fit against the SPR associationslope of the standard human fab of known concentration using CLAMP 3.40software to estimate the molar concentrations of each fab insupernatant.

Step 2: A high density goat anti-human fc polyclonal antibody surface(Invitrogen H10500) was prepared using standard amine coupling over twolanes of a GLC chip using a ProteOn XPR 36 biosensor. A high densityanti-mouse fc polyclonal antibody surface (GE Healthcare BR-1008-38) wasprepared using standard amine coupling over two different lanes of thesame GLC chip. The activation and blocking steps for all four capturesurfaces occurred in the vertical flow direction. Each fab insupernatant was then injected at three concentrations over fc-fusionhuman PVRIG (PVRIG-HH-2-1-1 #448, GenScript) and fc-fusion mouse PVRIG(PVRIG-MM-2-1-1 #198, GenScript) which were captured to one high densityanti-human fc surface and one anti-mouse fc surface (respectively) at anaverage of ˜200 RU and ˜290 RU per cycle, respectively. Each fabconcentration series was injected for two minutes followed by 10 minutesof dissociation at a flow rate of 50 μL/min. The starting concentrationrange (as determined in Step 1) was ˜20 nM-˜400 nM with two three-folddilutions of the highest concentration for each fab. Fabs were dilutedinto the running buffer which was degassed PBS with 0.05% Tween 20 and0.01% BSA added. The anti-human fc capture surfaces were regeneratedwith two 30-second pulses of 146 mM phosphoric acid after each cycle andthe anti-mouse fc surfaces were regenerated with two 30-second pulses of10 mM glycine, pH 1.7 after each cycle.

Step 3: Sensorgram data of fabs in supernatant binding to captured PVRIGwere processed and double-referenced using ProteOn Manager version3.1.0.6. The sensorgrams were double-referenced using the correspondinganti-species capture surfaces with no captured PVRIG as referencesurfaces and a blank injection over the captured PVRIG under identicalconditions as the injections of the fabs. Where possible, thesensorgrams for the three different concentrations of each fab were thenglobally fit to a 1:1 kinetic model (with a term for mass transport) toestimate the association and dissociation rate constants. Sensorgramswhich did not show simple 1:1 binding were not fit with the kineticmodel and therefore were not assigned estimates for ka and ka.

Results

None of the fabs included in this study showed binding activity to mousePVRIG (data not shown). Sensorgrams for 17 of the 50 fabs screenedagainst the human PVRIG could be fit for reliable estimates of theirrate constants. Twenty eight clones showed complex kinetics, five of thefabs did not show any binding to the captured human PVRIG fusion protein(CPA.7.025, CPA.7.026, CPA.7.027, CPA.7.029, CPA.7.035) and one clone(CPA.7.035) showed no titer when performing the concentrationdetermination in Step 1. The rate constants and their correspondingsensorgrams are shown below in FIG. 49 and FIGS. 50A-50Q. The cloneslisted below showed complex kinetics. FIGS. 51A-51C show some examplesof these data.

CPA.7.001 CPA.7.006 CPA.7.013 CPA.7.045 CPA.7.030 CPA.7.036 CPA.7.014CPA.7.046 CPA.7.031 CPA.7.037 CPA.7.041 CPA.7.017 CPA.7.032 CPA.7.009CPA.7.042 CPA.7.018 CPA.7.033 CPA.7.038 CPA.7.043 CPA.7.047 CPA.7.034CPA.7.039 CPA.7.016 CPA.7.023 CPA.7.003 CPA.7.011 CPA.7.044 CPA.7.024

Example 13 Measuring the Binding Affinity of IgG Clone CPA.7.021 toPVRIG Expressed on HEK Cells Using Flow Cytometry

Materials and Methods

Flow cytometry was used to measure the affinity of CPA.7.021 IgG bindingto human PVRIG expressed on HEK 293 cells. CPA.7.021 conjugated withAlexa 647 was added in duplicate at a binding site concentration rangeof 3 pM-101 nM in a 2-fold serial dilution to a constant number of cells(100,000 cells/well) over 17 wells in a 96-well plate. One wellcontained cells without any added IgG to serve as a blank well. Thecells were equilibrated for 4 hours with IgG at 4° C. Cells were washedtwice and then the Mean Fluorescence Intensity (MFI) was recorded overapproximately 10,000 “events” using an Intellicyte flow cytometer. Theresulting MFI values as a function of the CPA.7.021 IgG binding siteconcentration are shown below. The KD of CPA.7.021 binding to HEK 293cells expressing human PVRIG was estimated by fitting the MFI vs. theIgG binding site concentration curve with a 1:1 equilibrium model asdetailed in Drake and Klakamp, Journal of Immunol Methods, 318 (2007)147-152.

Results: Alexa647 labelled CPA.7.021 IgG was titrated with HEK 293 cellsexpressing human PVRIG and the binding signal was measured using flowcytometry. The resulting binding isotherm, showing MFI in duplicate vs.the binding site concentration of CPA.7.021, is presented below. The redline is a 1:1 equilibrium fit of the curve that allows for a KD estimateof 2.5 nM±0.5 nM (95% confidence interval of the fit, N=1).

Example 14 Effect of PVRIG Knock Down (KD) and Anti-PVRIG Antibody onHuman Melanoma Specific TILs Function

The aim of these assays is to evaluate the functional capacity of PVRIGin human derived TILs, as measured by activation markers and cytokinesecretion, upon co-culture with melanoma target cells. PD1 was used as abenchmark immune-checkpoint for the knock down (siRNA) studies. Theeffect of anti-PVRIG antibody (CPA.7.21), which has been shown to blockthe interaction of PVRIG and PVRL2, alone or in combination with otherantibodies (e.g aTIGIT, DNAM1) was evaluated.

Materials and Methods

TILs

Tumor-Infiltrating Lymphocytes (TILs) from Three Melanoma Patients wereUsed:

-   -   TIL-412-HLA-A2-Mart1 specific    -   TIL-F4-HLA-A2-gp100 specific    -   TIL-209-HLA-A2-gp100 specific

TILs were thawed in IMDM (BI, 01-058-1A) full medium supplemented with10% human serum (Sigma, H3667)+1% Glutamax (Life technologies,35050-038)+1% Na-Pyruvate (Biological Industries, 03-042-1B)+1%non-essential amino acids (Biological Industries, 01-340-1B)+1%Pen-Strep (Biological Industries, 03-031-1B)+300 U/ml of rhIL2(Biolegend, 509129).

Tumor cell lines: Human melanoma cells Mel-624 express MART-1 and gp-100antigens in the context of MHC-I haplotype HLA-A2. Cells were culturedin complete DMEM medium (Biological Industries, 01-055-1A) supplementedwith 10% FBS (BI, 04-127-1A), 25 mM HEPES buffer (BI, 03-025-1B), 1%Glutamax (Life technologies, 35050-038), and 1% Pen-Strep (BiologicalIndustries, 03-031-1B).

Knock down in TILs: Knock-down (KD) of human PVRIG and human PD1 in TILswas done using 100 pmol of Dharmacon ON-TARGETplus human PVRIGsiRNA-SMARTpool (L-032703-02) or Human PD1 siRNA—SMARTpool (L-004435) ornon-targeting siRNA (D-001810-01-05). siRNA were electroporated to TILs(AMAXA, program X-005). Electroporation was done on resting TILscultured in full IMDM supplemented with IL-2 24 hr post thawing. Afterthe electroporation TILs were seeded in 96 well TC plate to recover for24 hr. After 24 hr, cells were harvested and stained with viability dye(BD Horizon; Cat #562247, BD biosciences), washed with PBS and stainedwith anti-human PVRIG—CPA.7.021 (CPA.7.021 IgG2 A647, 7.5 μg/ml) or withanti-human PD-1 (Biolegend, #329910 AF647, 5 μg/ml) in room temperaturefor 30 min. isotype control used are synagis (IgG2 A647, 7.5 μg/ml) andmouse IgG1 (Biolegend #400130 A647, 5 μg/ml) respectively. All sampleswere run on a MACSQuant analyzer (Miltenyi) and data was analyzed usingFlowJo software (v10.0.8).

Co-culture of TILs with 624 melanoma cells: siRNA electroporated TILswere harvested and seeded in 96 TC plate 5×104/well. Mel-624 cells wereharvested as well and seeded in 1:1/1:3 E:T ratios in co-culture. Theplate was incubated overnight (18 hr) in 37° C., 5% CO2.

To assess the effect of anti-PVRIG antibody (CPA.7.021), anti-TIGIT(Clone 10A7) and anti-DNAM1 (clone DX11) on melanoma specific TILactivity, TILs (1×10⁵ cells/well) were pre-incubated with testedantibodies or relevant isotype controls in mono-treatment (10 μg/mL) orin combination-treatment (final 10 μg/mL for each) prior to the additionof 624 Melanoma target cells at a 1:1 Effector:target ratio. The platewas incubated overnight (18 hr) in 37° C., 5% CO2.

Assessment of TILs activation: 16 hours post co-culture, cells werestained with viability dye (BD Horizon; Cat #562247, BD biosciences),washed with PBS and exposed to Fc blocking solution (cat #309804,Biolegend), followed by surface staining with anti-CD8a (Cat #301048,Biolegend) and anti-CD137 (Cat #309804, Biolegend) in 4° C. for 30 min.All samples were run on a MACSQuant analyzer (Miltenyi) and data wasanalyzed using FlowJo software (v10.0.8). Culture supernatants werecollected and analyzed for cytokine secretion by CBA kit (Cat #560484,BD).

Results

PVRIG Knock-Down in TILs: TIL MART-1 and TIL F4 were cultured 24 hr withIL-2. 100 pmol of ON-TARGETplus human PVRIG siRNA—SMART pool(L-032703-02) or Human PD1 siRNA—SMARTpool (L-004435) or non-targetingsiRNA (D-001810-01-05) were electroporated to TILs (AMAXA, programX-005). Detection of PVRIG or PD-1 was performed 24 hr postelectroporation (and prior to co-culture). Cells were stained forviability dye followed by 30 min RT incubation with anti PVRIG or antiPD-1. The percentage of KD population is indicated in FIG. 82.

Functional assay using knocked down TILs: Human TILs, cultured for 24hours with IL2 were electroporated with siRNA encoding for human PVRIGor PD-1 or scrambled sequence as control. TILs were tested for PVRIG andPD-1 expression 24 hr post electroporation. ˜80% knock down of PVRIG and˜50% knock down of PD-1 compared to scrambled-electroporated TILs wasobserved (FIG. 82).

KD TILs were cultured with Mel-624 cells in 1:1 or 1:3 E:T for 18 hr andwere stained for the expression of CD137. Elevated levels of activationmarker CD137 were shown in TIL MART-1 electroporated with PVRIG siRNA,similarly to TILs that were electroporated with PD-1 siRNA, compared tocontrol scrambled siRNA (FIG. 83A). Co-culture supernatant was collectedand tested for the presence of secreted cytokines. TILs that wereelectroporated with PVRIG siRNA show a significant increase in IFNγ andTNF levels compared to control SCR siRNA. A similar effect was shown inTILs that were electroporated with PD-1 siRNA (FIG. 83B-83C).

The same trend of increase in activation levels was observed in TIL F4.Co-culture of PVRIG siRNA electroporated TIL F4 with Mel-624 in 1:3 E:Tled to increased levels of CD137 surface expression (FIG. 84A) as wellas increased secretion of IFNγ in co-culture supernatant (FIG. 84B).Similar trends were observed in TILs that were electroporated with PD-1siRNA.

Functional Assay Using Blocking Abs:

In vitro monotherapy and combo therapy of anti-PVRIG and anti-TIGIT: 209TILs were cultured with Mel-624 cells in 1:1 E:T for 18 hr. Co-culturesupernatant was collected and tested for the presence of secretedcytokines. Treatment with anti TIGIT did not affect IFNγ or TNFsecretion levels. However, an increase in IFNγ and TNF levels wasobserved when anti TIGIT and anti PVRIG were added to co-culture incombination (FIGS. 85A-85B).

In vitro monotherapy and combo therapy of anti-PVRIG and anti-TIGIT: 209TILs were cultured with Mel-624 cells in 1:1 E:T for 18 hr. TILs werestained for surface expression of activation marker CD137 and showedreduced level of expression upon treatment with anti DNAM-1. Co-culturesupernatant was collected and tested for presence of secreted cytokines.Treatment of anti DNAM-1 mediated a trend to increase secreted cytokinesIFNγ and TNF. Treatment with anti DNAM-1 and anti PVRIG in combinationpartially reversed the effect on CD137 expression (FIG. 86C) andenhanced the effect on cytokine secretion IFNγ and TNF (FIG. 5A-B).MART-1 TILs were cultured with Mel-624 cells in 1:1 E:T for 18 hr.Co-culture supernatant was collected and tested for the presence ofsecreted cytokines. Treatment with anti DNAM-1 reduced CD137 surfaceexpression on TILs and also the secreted cytokines IFNγ and TNF.Treatment with anti DNAM-1 and anti PVRIG in combination partiallyreversed these effects (FIGS. 86D-86F).

Summary and Conclusions

PD1 KD improved TIL activity, as measured by IFNγ and secretion in F4and MART-1 TILs. An increase (20%) of IFNγ and TNF secretion wasobserved upon PVRIG KD in MART-1 TILs compared to control siRNA. Thesame trend was observed in CD137 expression upon co-culture with 624Melanoma cells on F4 TILs.

Treatment of anti-TIGIT did not affect IFNγ or TNF secretion levels fromTILs co-cultured with 624 Mels, however, an increase in IFNγ and TNFlevels was observed when anti TIGIT and anti PVRIG (CPA.7.021) wereadded to co-culture in combination.

Anti DNAM-1 treatment reduced TIL-MART-1 activation manifested byreduced CD137 and cytokine secretion and anti-PVRIG (CPA.7.21) couldpartially reverse this effect in combo treatment with DNAM-1 Ab. In TIL209, IFNγ and TNF secretion levels were slightly elevated (10%) withanti DNAM-1, and an increase in IFNγ and TNF levels (40% and 30%,respectively) was observed when anti DNAM1 and anti PVRIG (CPA.7.021)were added to co-culture in combination. Collectively, our resultssuggest that PVRIG is a new co-inhibitory receptor for PVRL2.

Example 15 Effect of Anti-PVRIG Antibody on Human Melanoma Specific TilsFunction in Combination with Anti-TIGIT and Anti-PD1 Antibodies

Materials and Methods

TILs: Tumor-Infiltrating Lymphocytes (TILs) from Three Melanoma Patientswere Used:

1. TIL-412-HLA-A2-Mart1 specific

2. TIL-F4-HLA-A2-gp100 specific

3. TIL-209-HLA-A2-gp100 specific

TILs were thawed in IMDM (BI, 01-058-1A) full medium supplemented with10% human serum (Sigma, H3667)+1% Glutamax (Life technologies,35050-038)+1% Na-Pyruvate (Biological Industries, 03-042-1B)+1%non-essential amino acids (Biological Industries, 01-340-1B)+1%Pen-Strep (Biological Industries, 03-031-1B)+300 U/ml of rhIL2(Biolegend, 509129).

Tumor cell lines: Human melanoma cells Mel-624 express MART-1 and gp-100antigens in the context of MHC-I haplotype HLA-A2. Cells were culturedin complete DMEM medium (Biological Industries, 01-055-1A) supplementedwith 10% FBS (BI, 04-127-1A), 25 mM HEPES buffer (BI, 03-025-1B), 1%Glutamax (Life technologies, 35050-038), and 1% Pen-Strep (BiologicalIndustries, 03-031-1B).

Co-culture of TILs with 624 melanoma cells in the presence ofanti-PVRIG, anti-TIGIT and PD1 blocking antibodies: To assess the effectof anti-PVRIG antibody (CPA.7.021), anti-TIGIT (Clone 10A7) and anti-PD1(mAb 1B8, Merck) on melanoma specific TIL activity, TILs (3×104cells/well) were pre-incubated with tested antibodies or relevantisotype controls in mono-treatment (10 μg/mL) or incombination-treatment (final 10 μg/mL for each) prior to addition of 624Melanoma target cells at 1:3 Effector:target ratio. Plate was incubatedovernight (18 hr) in 37° C., 5% C02.

Assessment of TILs activation: Culture supernatants were collected andanalyzed for cytokine secretion by CBA kit (Cat #560484, BD).

In vitro monotherapy anti-PVRIG and combo-therapy of with anti-TIGIT andPD1 blocking antibodies: F4 TILs (gp100 specific) were cultured withMel-624 cells in 1:3 E:T for 18 hr. Co-culture supernatant was collectedand tested for presence of secreted cytokines. Treatment of anti-TIGITor anti-PD1 did not affect IFNγ or TNF secretion levels. However, anincrease in IFNγ and TNF levels was observed when anti TIGIT or anti-PD1in combination with anti PVRIG were added to co-culture in combination(FIGS. 87A-87B).

Treatment of anti-PVRIG, anti-TIGIT and PD1 alone did not affect IFNγ orTNF secretion levels from TILs co-culture with 624 Mels, however, anincrease in IFNγ and TNF levels was observed when anti-TIGIT or anti-PD1antibodies were added in combination with anti PVRIG (CPA.7.021). Thepresented data suggest that there is synergestic effect for combinatorytherapy with anti-TIGIT or anti-PD1 antibodies.

Example 16: Effect of Anti-PVRIG Antibodies on TCR Signaling UsingReporter Gene Assay

A reporter assay system for TCR signaling, such as the Jurkat-NFAT-Luccell line, is used to test the effect of anti-PVRIG antibodies on TCRmediated signaling. This Jurkat cell line derivative expresses theluciferase reporter gene under the control of the NFAT response element.These cells are transfected with a vector encoding full length humanPVRIG. As negative control, cells transfected with empty vector areused. Transfectants with vectors encoding for costimulatory orcoinhibitory reference molecules, such as CD28 and PD-1, serve aspositive control. Transfectants are stimulated by the addition ofanti-human CD3 (e.g. OKT3) in the absence or presence of anti-PVRIGantibodies. Isotype control serves as negative control. Known functionalantibodies against the reference molecules serve as positive controls. Afunctional agonistic crosslinking antibody is expected to show aninhibitory effect on the luciferase activity.

Example 17 Effect of Anti-PVRIG Antibodies on T Cell Activation UsingPVRL2-Fc

A plate bound assay is used to test the effect of anti-PVRIG antibodieson T cell activation, proliferation and cytokine secretion. Purifiedhuman bulk T cells are stimulated using 1 μg/ml plate bound anti-humanCD3 (e.g. OKT3) and 5 μg/ml PVRL2-Fc (recombinant fused protein composedof the ECD of PVRL2, the counterpart of PVRIG) or negative control. Tcell activation is evaluated by expression of activation markers, e.g.CD137, or by cell division as evaluated by dilution of CFSE dye (T cellsare labeled with CFSE prior to their stimulation). Cytokine production(e.g. IFNg, IL-2) is also assessed as additional readout of T cellactivation. T cell subtype markers are used to distinguish specificeffects on CD4 or CD8 T cells. The co-immobilized PVRL2-Fc could have abasal stimulatory effect on T cell activation, mediated throughendogenous DNAM1—a known costimulatory counterpart receptor of PVRL2 onT cells. In the presence of antagonistic anti-PVRIG Abs, thisstimulatory basal effect of PVRL2-Fc is expected to be further enhanced,due to their blocking of the inhibitory influence of endogenous PVRIG onT cell activation. Accordingly, agonistic anti-PVRIG Abs are expected toshow inhibition of T cell activation.

Example 18: Effect of Anti-PVRIG Antibodies on T Cell Activation UsingPVRL2 Ectopic Expressing Cells

A cell based assay is used to test the effect of anti-PVRIG antibodieson T cell activation, proliferation and cytokine secretion. Purifiedhuman bulk or CD4 or CD8 T cells are stimulated upon co-culture with CHOstimulator cells (CHO cells expressing membrane-bound anti-CD3)ectopically expressing PVRL2 or empty vector. T cell activation isevaluated by expression of activation markers, e.g. CD137, or by celldivision as evaluated by dilution of CFSE dye (T cells are labeled withCFSE prior to their stimulation). Cytokine production (e.g. IFNγ, IL-2)is also assessed as additional readout of T cell activation. T cellsubtype markers are used to distinguish specific effects on CD4 or CD8 Tcells. The PVRL2-expressing CHO stimulators are expected to have a basalExample 19 stimulatory effect on T cell activation, mediated throughendogenous DNAM1—a known costimulatory counterpart receptor of PVRL2 onT cells. In the presence of antagonistic anti-PVRIG Abs, thisstimulatory basal effect of surface expressed PVRL2 is expected to befurther enhanced, due to their blocking of the inhibitory influence ofendogenous PVRIG on T cell activation. Accordingly, agonistic anti-PVRIGAbs are expected to show inhibition of T cell activation.

Example 20 Effect of Anti-PVRIG Antibodies on T Cell Activation Usingthe SEB Assay

Anti-PVRIG antibodies are tested for their effect on T cell activityusing blood cells from healthy volunteers and SEB (Staphylococcusenterotoxin B) superantigen to engage and activate all T cellsexpressing the V03 and V08 T cell receptor chain. Human PBMCs arecultured in 96-well round-bottom plates and pre-incubated for 30-60 minwith the tested antibodies. SEB is then added at various concentrationsranging from 10 ng/mL to 10 μg/mL. Supernatants are collected after 2 to4 days of culture and the amount of cytokine (e.g. IL-2, IFNγ) producedis quantified by ELISA or using standard CBA kit. SEB stimulatescytokine production by whole-blood cells in a dose dependent manner. Theeffect of anti-PVRIG mAbs on cytokine production is tested at several Abdoses. Blocking anti-PVRIG mAbs are expected to enhance IL-2 productionover control IgG. In addition to IL-2, the effect of the Abs on thelevels of additional cytokines such as TNFα, IL-17, IL-7, IL-6 and IFNγcan be tested in this assay using a CBA kit.

Example 21 Effect of Anti-PVRIG Antibodies in Ag-Specific Assays

An assay that is used to profile the functional effect of anti-humanPVRIG antibodies on Ag specific stimulation of pre-existing memory Tcells in healthy donor blood is the tetanus toxoid (TT) assay. To thisend, freshly prepared PBMC (2×105 cells) are plated in 96 wellround-bottom plates in complete RPMI 1640 medium (containing 5% heatinactivated human serum), pre-incubated with tested antibodies atvarying concentration and stimulated with TT (Astarte Biologics) at aconcentration of 100 ng/mL The cells are incubated for 3-7 days at 37°C., after which supernatants are harvested. Cytokine concentrations(e.g. IL-2, IFN-γ) are determined by ELISA and/or CBA kit. Blockinganti-PVRIG Abs are expected to enhance T cell proliferation and cytokineproduction compared to that obtained with TT antigen alone.

Similarly to the method described above, which uses TT to stimulatehuman memory T cells, we can test the effect of anti-PVRIG Abs on T cellactivation upon recall responses to additional antigens such as CMV,EBV, influenza HIV, mumps, and TB, using a similar experimental setup asdescribed above. This can also be used to test the effect of anti-PVRIGantibodies on stimulation of naïve cells using neo-antigens such as KLH.

In addition, the effect of anti-PVRIG Abs is tested on the antigenspecific responses of tetramer-sorted Ag-specific CD8 T cells fromperipheral blood of patients suffering from viral infections such as HCVand HIV. Tetramer sorted CD8 T cells are co-cultured with peptide-loadedautologous PBMCs for 5 days. Proliferation of CD8 Ag-specific T cellsand secretion of cytokines (e.g. IFNγ, IL2, TNF-α) are evaluated. Weexpect anti-PVRIG antibodies to enhance proliferation and cytokineproduction, compared to antigen alone.

Example 22 Binding and Functional Analysis of Hybridoma-DerivedAntibodies Against PVRIG

This example shows the characterization of binding of hybridoma-derivedantibodies (the CHA antibodies) to human and cynomolgus PVRIG protein incell lines and primary leukocytes, as well as the characterization ofthe capacity of hybridoma-derived antibodies to block the interactionbetween PVRIG and PVRL2.

Protocols

FACS analysis of hPVRIG over-expressing cells: The following cell lineswere used to assess the specificity of anti-human PVRIG antibodies: HEKparental and HEK hPVRIG over-expressing cells. These cells were culturedin DMEM (Gibco)+10% fetal calf serum (Gibco)+ glutamax (Gibco). For theHEK hPVRIG over-expressing cells, 0.5 μg/ml puromycin (Gibco) was alsoadded to the media for positive selection. For FACS analysis, all celllines were harvested in log phase growth and 50,000-100,000 cells perwell were seeded in 96 well plates. Anti-human PVRIG antibodies (mIgG1or mIgG2a) and their respective controls were added in single pointdilutions (5 μg/ml), or as an 8 point titration series starting at 10μg/ml on ice for 30 mins-1 hr. The titration series were conducted aseither 1:3 or 1:3.3 fold serial dilutions. Data was acquired using aFACS Canto II (BD Biosciences) or IntelliCyt (IntelliCyt Corporation)and analyzed using FlowJo (Treestar) and Prism (Graphpad) software.

FACS analysis of human cell lines for hPVRIG: The following cell lineswere used to assess the expression and specificity of anti-human PVRIGantibodies: Jurkat and HepG2. Jurkat cells were cultured in RPMImedia+10% fetal calf serum, glutamax, non-essential amino acids (Gibco),sodium pyruvate (Gibco), and penicillin/streptomycin (Gibco). HepG2cells were cultured in DMEM+10% fetal calf serum+ glutamax. For FACSanalysis, all cell lines were harvested in log phase growth and50,000-100,000 cells per well were seeded in 96 well plates. Anti-humanPVRIG antibodies (mIgG1 or mIgG2a) and their respective controls wereadded in single point dilutions (5 μg/ml), or as an 8 point titrationseries starting at 10 μg/ml on ice for 30 mins-1 hr. The titrationseries were conducted as either 1:3 or 1:3.3 fold serial dilutions. Datawas acquired using a FACS Canto II or IntelliCyte and analyzed usingFlowJo and Prism software.

FACS analysis of naïve human primary leukocytes for hPVRIG: Primaryleukocytes were obtained by Ficoll (GE Healthcare) gradient isolation ofperipheral blood (Stanford Blood Bank). Leukocytes as isolatedperipheral blood mononuclear cells (PBMC) were frozen down in liquidnitrogen at a density between 1×10⁷ and 5×10⁷ cells/ml in a 10% DMSO(Sigma), 90% fetal calf serum mixture. To assess protein expression ofPVRIG on PBMC, antibody cocktails towards major immune subsets weredesigned that included human anti-PVRIG antibodies. Anti-human PVRIGantibodies (mIgG1 or mIgG2a) and their respective controls were added insingle point dilutions (5 μg/ml), or in some cases, as a 4 pointtitration series starting at 10 μg/ml on ice for 30 mins-1 hr.

Briefly, antibody cocktail mixtures were added to resuscitated PBMC thatwere seeded at 5×10⁵-1×10⁶ cells/well upon prior Fc receptor blockadeand live/dead staining (Aqua Live/Dead, Life Technologies). Antibodycocktails were incubated with PBMC for 30 mins-1 hr on ice. PBMC werethen washed and data was acquired by FACS using a FACS Canto II. Datawas analysed using FlowJo and Prism software. Immune subsets that wereanalysed include CD56 dim NK cells, CD56 bright NK cells, CD4+ T cells,CD8+ T cells, non-conventional T cells (e.g. NKT cells and γδ T cells),B cells, and monocytes.

FACS analysis of cynomolgus PVRIG engineered over-expressing cells: Thefollowing cell lines were used to assess the cross-reactivity ofanti-human PVRIG antibodies with cynomolgus PVRIG (cPVRIG): expiparental and expi cPVRIG over-expressing cells. These cells werecultured in DMEM+10% fetal calf serum+ glutamax. expi cPVRIG transientover-expressing cells were generated by electroporating cPVRIG DNA intoparental expi cells using the Neon transfection system. For FACSanalysis, expi cPVRIG cells were used between 1-3 days posttransfection. Parental expi cells were harvested from log growth phase.50,000-100,000 cells of per well of each type were seeded in 96 wellplates. Anti-human PVRIG antibodies (mIgG1 or mIgG2a) and theirrespective controls were added in single point dilutions (5 μg/ml), oras an 8 point titration series starting at 10 μg/ml on ice for 30 mins-1hr. The titration series were conducted as either 1:3 or 1:3.3 foldserial dilutions. Data was acquired using a FACS Canto II or IntelliCyteand analyzed using FlowJo and Prism software.

FACS analysis of naïve primary cynomolgus monkey leukocytes: Primarycynomolgus monkey (cyno) leukocytes were obtained from fresh blood whichwas drawn no longer than 24 hours prior to expression analysis. Bloodwas sourced from Bioreclamation. To assess protein expression of PVRIGon cyno PBMC, antibody cocktails towards major immune subsets weredesigned that included human anti-PVRIG antibodies. Anti-human PVRIGantibodies (mIgG1 or mIgG2a) and their respective controls were added insingle point dilutions (5 μg/ml), or as an 8 point titration seriesstarting at 10 μg/ml on ice for 30 mins-1 hr.

Briefly, antibody cocktail mixtures were added to PBMC that were seededat 5×10⁵-1×10⁶ cells/well upon prior Fc receptor blockade and live/deadstaining. Antibody cocktails were incubated with PBMC for 30 mins-1 hron ice. PBMC were then washed and data was acquired by FACS using a FACSCanto II. Data was analysed using Prism software. Immune subsets thatwere analysed include CD16+ lymphocytes, CD14+/CD56+ monocytes/myeloidcells, and CD3+ T cells.

Cellular-based competition assays: The ability of PVRIG antibodies toinhibit the interaction of PVRIG with its ligand PVRL2 was assessed in acellular competition assay. In this assay, the ligand PVRL2 isendogenously expressed on un-manipulated HEK cells and soluble Fc-taggedPVRIG (manufactured on demand by Genscript) is added. In this case, theability of PVRIG antibodies to block soluble PVRIG binding to HEK cellswere assessed through the concomitant addition of 33 nM of soluble PVRIGprotein and PVRIG antibodies (0.066-66 nM) to 100,000 HEK cells andincubated for 1 hour on ice. The extent of PVRIG Fc binding was detectedby addition of anti-human Fc Alexa 647 (Jackson Laboratories) for 20-30minutes on ice. Cells were washed twice in PBS for acquisition using aFACS Canto II. Data was analyzed using FlowJo (Treestar), Excel(Microsoft) and Prism (GraphPad).

Results

Hybridoma PVRIG antibodies recognize PVRIG on overexpressing cells: Toscreen for antibodies that were specific for PVRIG, we assessed theability of antibodies that were generated from two hybridoma campaignsto bind HEK cell lines that were engineered to overexpress human PVRIG.The majority of antibodies from these campaigns bound to the HEK hPVRIGcells, albeit with varying affinity. Furthermore, the majority of theseantibodies also showed low background binding to HEK parental cell linesindicating high specificity towards PVRIG. FIG. 77 shows one example ofthe specificity of the PVRIG antibodies. A summary of all bindingcharacteristics of the antibodies towards HEK hPVRIG cells relative tocontrol that were generated in the hybridoma campaigns are displayed inFIGS. 79A-79B.

PVRIG antibodies recognize PVRIG protein on naïve NK and T cells: Thepopulations which displayed the highest level of PVRIG on naïve PBMCsubsets were NK and CD8 T cells, and the absolute level of expressionbetween these two cell subsets was similar (gMFI). CD4 T cells showedlower levels of PVRIG, while B cells and monocytes had very low/nodetectable expression. A summary of expression on naïve NK cells and CD8T cells as detected by the antibodies is shown in FIG. 91. Other minorsubsets also displayed PVRIG expression and included non-conventional Tcells such as NKT cells and γδ T cells. The expression pattern on PBMCsubsets was very similar across all donors sourced and analyzed.

PVRIG is detected on Jurkat cell lines by hybridoma-derived PVRIGantibodies: In addition to screening PBMC for PVRIG protein expression,we wanted to understand whether it was also expressed on cancer celllines. We chose to screen our antibodies on Jurkat cells given theirhigh expression of PVRIG RNA. We also chose HepG2 as a negative controlcell line to further validate the specificity of our antibodies. Most ofthe hybridoma-derived antibodies did detect PVRIG protein expression onJurkat cells (FIGS. 79A-79B PVRIG hybridoma antibody bindingcharacteristics to primary human PBMC, cyno over-expressing cells, andcyno primary PBMC. Expi cyno OE denotes expi cells transientlytransfected with cPVRIG, expi par denotes expi parental cells. gMFIrindicates the fold difference in geometric MFI of PVRIG antibodystaining relative to their controls. Concentrations indicate that atwhich the gMFIr was calculated. Not tested indicates antibodies thatwere not tested due to an absence of binding to human HEK hPVRIG, expicPVRIG cells, or not meeting binding requirements to PBMC subsets.Highlighted antibodies are four antibodies for which humanization wasdone (See FIG. 90).

FIG. 92), but not the HepG2 cells (data not shown). An example of PVRIGdetection on Jurkat is shown in FIG. 78 with a representative antibody,CHA.7.518.

Cellular-based biochemical assays Upon screening our 29 hybridomaantibodies in the cellular biochemical assays, we found that there were20 clear blockers and 9 non-blockers of the PVRIG-PVRL2 interaction. Allof the blocking antibodies were able to inhibit the interaction of PVRIGFc with HEK cells by at least 50%, with most of these antibodiescompletely abolishing PVRIG Fc binding. The IC₅₀ values associated withthose antibodies that did show blocking capacity are reported in FIG.92. The majority of IC₅₀ values were between 20-60 nM.

Summary and Conclusions

Using a hybridoma platform, we have been able to successfully generatemonoclonal antibodies towards the human PVRIG antigen. Using engineeredover-expressing cells as well as a suite of cancer cell lines, we showedthat our antibodies are highly specific to the PVRIG antigen, and areable to detect protein expression which correlated with RNA expression.Upon analysis of human PBMC subsets, we showed that the PVRIG protein ismost highly expressed on NK and T cells, with low/negative expression onB cells and myeloid cells. We also showed that a proportion of theseantibodies are cross-reactive with the cynomolgus monkey (cyno) PVRIGantigen through assessing their binding to over-expressing cells.Furthermore, the expression pattern on cyno PBMC is similar to humanPBMC. Lastly, we were able to show through a FACS-based competitionassay, that a proportion of our hybridoma antibodies are able to inhibitthe interaction of PVRIG with its ligand, PVRL2. The antibodies whichshowed the best characteristics regarding all the aforementioned datawere CHA-7-518, CHA-7-524, CHA-7-530, and CHA-7-538.

Example 23. Effect of CHA Anti-PVRIG Antibodies in the MLR Assay

An assay used to profile the functional effect of anti-human PVRIGantibodies on allo-antigen responses is proliferation of Human CD8+ TCells in a Mixed Lymphocyte Reaction (MLR) assay. As is known in theart, MLR is an ex vivo cellular immune assay that provides an in vitrocorrelation of T cell function.

Anti-PVRIG antibodies are expected to enhance proliferation of human CD4and CD8 T cells in response to cells from an MHC-mismatched donor. HumanT cells are enriched from whole blood of one donor (e.g. donor A) byusing Human T cell RosetteSep® (StemCell Technologies) as permanufacturer's instructions. After separation, cells are fluorescentlylabeled with CFSE dye (Molecular Probes). To serve as allogeneic antigenpresenting cells (APCs), mononuclear cells are first isolated from wholeblood from a MHC-mismatched donor (e.g. donor B) and then depleted ofCD3+ T cells. APCs are then irradiated with 2500 rads in a cesiumirradiator.

In general, an MLR assay is done as follows. HumanT cells and allogeneic150,000 APCs are co-cultured in a 96-well flat-bottom plate with 150,000CD8+ T cells and APCs for 5 days with anti-PVRIG antibodies at differentconcentrations. On day 5, cells are harvested, washed and stained withanti-CD8-biotin followed by streptavidin-PerCp. Samples are run by FACSto assess the degree of proliferation as depicted by CFSE dilution.Functional blocking anti-PVRIG antibodies are expected to enhance Tcells proliferation and cytokine secretion in response to cells from aMHC-mismatched donor.

An MLR assay was used to characterize the biochemical effect of the CHAantibodies of the invention on resting and activated human T cells, andto characterize the capacity of hybridoma-derived antibodies to modulateT cell proliferation in an MLR setting

Protocols

Mixed Lymphocyte Reaction (MLR): A mixed lymphocyte reaction wasestablished by co-culturing dendritic cells (DCs) and T cells derivedfrom distinct donors in an allogeneic setting. DCs were generated byculturing purified monocytes with 100 ng/ml GM-CSF (R&D systems) and 100ng/ml IL-4 (R&D systems) for 7 days. After 7 days, purifiedCFSE-labelled CD3 T cells were combined with DCs at a 10:1 ratio andwere cultured in X vivo-20 serum free media (Lonza) for 5 days. In someconditions, unconjugated anti-PVRIG antibodies or isotype controlantibodies were added to the plates at 10 μg/ml. Three MLR assaypermutations were set up, where DCs from one donor were co-cultured withCD3 T cells from 3 separate allogeneic donors. All blood products weresourced from Stanford Blood Bank.

Expression and functional analysis: After the 5 day MLR culture, thelevel and extent of T cell activation and proliferation was assessed byCFSE dilution and expression of activation markers such as CD25 andPD-1. In-house anti-PVRIG antibodies from both phage and hybridomacampaigns were used to assess the expression of PVRIG. Expression of thePVRIG ligand, PVRL2, was also assessed in a kinetic fashion on DC. Alldata was acquired using flow cytometry and data analysis was performedusing FlowJo (Treestar) and Prism (Graphpad) software.

FACS-based epitope analysis: As we tested an array of antibodies in theMLR, we were interested in determining whether these antibodies could beepitope ‘binned’ based on FACS-based binding, and whether this ‘binning’would correlate to changes in T cell activation and proliferation in theassay. To do this, T cells harvested from the assay were pre-incubatedwith unconjugated PVRIG antibodies, and then counter-stained with aconjugated PVRIG antibody of a different clone. The extent to which theconjugated PVRIG antibody gave a signal on T cells indicated the extentto which this antibody had to compete for PVRIG binding on T cells withthe unconjugated antibody. A negative or low signal would indicate thatthere is high competition, indicating the two antibodies are in the sameepitope ‘bin’. A high signal would indicate low or no competition andthus the antibodies would be considered to be in different ‘bins’.

Results

Expression of PVRL2 on monocyte-derived DC: To determine whether PVRL2would be expressed on DC for the MLR assay, DC were generated frommonocytes, and PVRL2 expression was assessed in a kinetic fashion atdaily intervals after addition of GM-CSF and IL-4. As indicated in FIG.72, PVRL2 expression increased from Day 0 until Day 5 where expressionpeaked. At Day 6, expression decreased slightly compared to Day 5. AtDay 7, expression was similar to Day 6 indicating stabilization of PVRL2expression at these time points. Thus, DC expressed PVRL2 at theappropriate time point for use in the MLR assay.

Expression of PVRIG on T cells after MLR culture: Many T cell receptorsthan modulate function in the MLR are expressed on proliferating Tcells. Thus, we wanted to determine whether PVRIG is also expressed. Weanalysed proliferating T cells at Day 5 post MLR co-culture initiationand were characterized by their dilution of CFSE (i.e. CFSE low). Asshown in FIGS. 73A-73B and FIGS. 74A-74B, relative to isotype control(mIgG1), PVRIG was expressed on CFSE low cells as determined by multiplePVRIG antibodies on both CD4 and CD8 T cells across three donorsanalysed. FACS plots are shown in FIGS. 73A-73B to indicate PVRIG onCFSE low cells, and bar graphs in FIGS. 74A-74B indicate the level ofexpression of PVRIG relative to mIgG1.

PVRIG antibodies enhance T cell proliferation: Having shown that PVRIGexpression is expressed on proliferating T cells in the MLR, we wantedto determine whether treatment with PVRIG antibodies could affect levelsof T cell proliferation. As shown in FIG. 4, addition of PVRIGantibodies into the MLR assay was able to increase the percentage ofCFSE low cells across all the hybridoma antibodies tested compared tocontrol. This was observed across all donors analysed.

PVRIG antibodies bind to multiple epitopes on PVRIG: To compare thePVRIG antibodies for their ability to bind different epitopes on PVRIG,we performed a competition experiment where T cells from the MLR werecultured with unlabeled anti-PVRIG antibodies derived from our hybridomacampaigns for 5 days. T cells were then harvested at day 5 andcounter-stained with a conjugated anti-PVRIG antibody that was derivedfrom our phage campaign (CPA.7.021). As shown in FIG. 76, complete ornear complete reduction of CPA.7.021 binding was observed in conditionsthat contained CHA.7.516-M1, CHA.7.518-M1, CHA.7.524-M1, CHA.7.530-M1,and CHA.7.538-M1 when compared to background fluorescence levels,suggesting that these antibodies may overlap in epitope recognition.Partial reduction in CPA.7.021 binding was observed with CHA.7.537-M1,CHA.7.528-M1, and CHA.7.548-M1, suggesting partial overlap in epitoperecognition. No reduction in CPA.7.021 binding was observed in cellspre-cultured with CHA.7.543-M1 suggesting an absence of epitoperecognition. Collectively, this data indicates that the PVRIG antibodiesfrom our campaigns, when assessed relative to CPA.7.021, could recognizeat least 3 different epitopes on PVRIG.

Conclusions

We characterized our PVRIG antibodies for their ability to bind toproliferating and resting T cells, as well as their functional activityin a MLR. Binding of multiple PVRIG antibodies was detected onproliferating T cells and was higher on proliferating T cells ascompared to resting, especially the CD8+ subset. This data demonstratesthat PVRIG expression is increased upon T cell activation. Furthermore,several PVRIG antibodies increased T cell proliferation as compared tomIgG1 isotype indicating that they can also modulate T cell function. Asabove, these antibodies all have ability to block PVRIG with its ligand,PVRL2. Based on this, we conclude that by blocking the PVRIG-PVRL2interaction, these antibodies lead to an increase in T cell activationand proliferation, which is a hallmark indication of a desired effectfor an immune checkpoint inhibitor that would be used to treat cancer.Lastly, we performed competition experiments comparing the binding ofmultiple hybridoma-derived PVRIG antibodies to activated T cells,relative to a phage-derived antibody. From this series of experiments,we provide evidence for epitope diversity of our phage andhybridoma-derived antibodies.

Example 24. Effect of Anti-PVRIG Antibodies on T Cell Activation UponCombination with Immune Checkpoint Blockade

The combination of PVRIG blockade with blocking Abs of a known immunecheckpoint (e.g. PD1, PDL-1 or TIGIT), is expected to further enhancethe stimulatory effect on T cell activation in the assays depictedabove.

Example 25. Functional Analysis of PVRIG Antibodies

The human PVRIG antibodies of the invention were characterized for theability to inhibit the interaction of PVRIG with its ligand PVRL2, andtheir ability to modulate effector lymphocyte function in primarycell-based assays.

Protocols

Cellular-Based Biochemical Assays

The ability of PVRIG antibodies to inhibit the interaction of PVRIG withits ligand PVRL2 was assessed in a cellular biochemical assay format intwo orientations.

In the first orientation, the ligand PVRL2 is endogenously expressed onun-manipulated HEK cells and soluble biotinylated Fc-tagged PVRIG(manufactured on demand by Genscript) is added. In this case, theability of PVRIG antibodies to block soluble PVRIG binding to HEK cellswere assessed through two permutations. In the first permutation,various concentrations of PVRIG antibodies (range 0.066-66 nM) werepre-incubated with 33 nM of soluble PVRIG in phosphate buffered saline(PBS, Gibco) for 30 minutes on ice. This complex was subsequently addedto 100,000 HEK cells in and incubated for a further 1 hour on ice. After1 hour, HEK cells were washed twice in PBS and the extent of solublePVRIG bound to HEK cells was detected by addition of streptavidinconjugated to Alexa 647 (Jackson Laboratories) for 30 minutes on ice.HEK cells were washed twice in PBS, and resuspended in 100 ul of PBS foracquisition on the FACS Canto II (BD Biosciences). Data was analysedusing FlowJo (Treestar) and Prism (Graphpad) software. In the secondpermutation, 33 nM of soluble PVRIG protein and PVRIG antibodies(0.066-66 nM) were added concomitantly to 100,000 HEK cells andincubated for 1 hour on ice. Subsequent steps to analysis for thispermutation are equivalent to the first permutation.

In the second orientation, HEK cells were engineered to over-expressPVRIG and soluble biotinylated Fc-tagged PVRL2 (CD Biosciences) wasadded. In this case, various concentrations of PVRIG antibodies (range0-200 nM) with 160 nM soluble PVRL2 were added concomitantly to 100,000HEK hPVRIG or parental HEK cells, and incubated in PBS +1% BSA+0.1%sodium azide (FACS buffer) for 1 hr on ice. Soluble PVRL2 binding wasdetected by addition of streptavidin Alexa 647 in FACS buffer for 30minutes on ice. Cells were washed twice in FACS buffer, and re-suspendedin 50 ul of PBS for acquisition on the Intellicyt HTFC (Intellicyt).Data was analyzed using FlowJo (Treestar), Excel (Microsoft) and Prism(GraphPad).

Primary NK Cell Assay

The PBMC subset with the most robust expression profile for PVRIG was onNK cells. As such, we designed an NK cell-based co-culture assay withPVRL2-expressing tumor cells to determine whether our antibodies couldmodulate NK cell-mediated cytotoxicity towards these targets. Thetargets we chose were the acute B cell lymphocytic leukemia cell line,Reh (ATCC cell bank), and the acute myeloid leukemia cell line, MOLM-13(DSMZ cell bank). Reh and MOLM-13 cells were grown in RPMI media(Gibco)+20% fetal calf serum (Gibco), glutamax (Gibco),penicillin/streptomycin (Gibco), non-essential amino acids (Gibco),sodium pyruvate (Gibco), HEPES (Gibco), and beta-mercaptoethanol(Gibco).

Two days prior to the co-culture assay, primary NK cells were isolatedusing the human NK cell isolation kit (Miltenyi Biotec) and cultured inRPMI media+20% fetal calf serum, glutamax, penicillin/streptomycin,non-essential amino acids, sodium pyruvate, HEPES, beta-mercaptoethanol,and 250 U/ml IL-2 (R&D systems). On the day of the assay NK cells wereharvested, enumerated and pre-incubated with PVRIG antibodies for 15-30minutes at room temperature. During this incubation, target cells wereharvested from culture, labelled with Calcein AM (Life Technologies) for30 minutes at 37° c., washed in media, and enumerated for the assay. NKcell-mediated cytotoxicity assays were set up where a constant number oftarget cells (50,000) were co-cultured with increasing concentrations ofNK cells pre-incubated with 5 μg/ml of PVRIG antibodies (thus alteringthe NK cell to target ratio). Alternatively, a fixed NK cell to targetratio was used in the assay, but NK cells were pre-incubated withaltering concentrations of PVRIG antibody (range 3.9 ng/ml-5 μg/ml) in adose titration. Upon addition of the NK cells and targets, plates werepulse spun at 1,400 rpm for 1 minute and placed at 37° c. in a 5% CO2atmosphere for 4 hours. After 4 hours, plates were spun at 1,400 rpm for4 minutes, and 80 ul of supernatant was harvested to quantitate therelease of Calcein AM from the target cells. The quantity of Calcein AMreleased from targets was assessed by a Spectramax Gemini XS fluorometer(Molecular Devices). As controls for Calcein AM release, total andspontaneous release was assessed by exposing target cells to 70% ethanolor media only for the duration of the assay. Levels of killing (as apercentage) by NK cells were calculated using the following formula:

(Sample release −spontaneous release)/(total release −spontaneousrelease)*100

In addition to PVRIG antibodies, in some cases, other antibodies towardsNK cell receptors such as TIGIT (Genentech, clone 10A7, Patent number:WO2009126688 A2) and DNAM-1 (Biolegend, clone 11A8) were also added ascomparators.

Results

Cellular-based biochemical assays: Upon screening a panel of our PVRIGantibodies in the cellular biochemical assays, we found that there wasvariable levels of inhibition across the antibodies tested, and thelevel of inhibition was dependent on the permutation and orientation ofthe assay (FIG. 98). Four antibodies are specifically shown in FIGS.93A-93C to illustrate these points. The orientation and permutation ofthe assay which gave the most robust inhibitory effect relative tocontrol, was when soluble PVRIG pre-incubated with PVRIG antibodies wasadded to HEK cells (FIG. 93a ). In this permutation, CPA.7.021 showedthe best absolute blocking capacity compared to the other threeantibodies (CPA.7.002, CPA.7.005, and CPA.7.050). Despite thedifferences in level of blocking, all antibodies in this permutationshowed similar IC₅₀ values which were in the low nanomolar range, andthe blocking capacity plateaued at higher concentrations.

When the absolute level of inhibition invoked by the four PVRIGantibodies was then measured when soluble PVRIG and PVRIG antibodieswere concomitantly added to HEK cells, more variability of blocking inthe assay was observed (FIG. 93b ). CPA.7.021 remained the best blockingantibody. However, CPA.7.002 and CPA.7.005 showed markedly less abilityto inhibit soluble PVRIG binding to HEK cells relative to the controlantibody. CPA.7.050 showed an intermediate level of blocking as comparedto CPA.7.021, CPA.7.002, and CPA.7.005. This difference in absolutelevel of inhibition also corresponded to differences in the IC₅₀ valuesof each antibody. CPA.7.021 and CPA.7.050 again showed low nanomolarIC₅₀ values, although they were both higher than in the firstpermutation of the assay. In contrast, the IC₅₀ values of CPA.7.002 andCPA.7.005 increased substantially, CPA.7.002 by approximately 20-fold,and CPA.7.005 by approximately 30-fold. This data indicates that how theantibody has to compete for PVRIG binding with its cognate ligand, willindicate the potency with which the antibody can block this interaction.

When the orientation of the biochemical assay was reversed (i.e. PVRL2Fcwas assessed to bind to HEK hPVRIG cells), the ability of the four PVRIGantibodies to block PVRL2 Fc interaction was variable (FIG. 93c ).Consistent with the biochemical assays which used HEK cells as targets(FIGS. 93a-b ), CPA.7.021 and CPA.7.050 inhibited PVRL2 Fc binding toHEK hPVRIG cells, and their ability to block the binding was similar.Surprisingly however, we saw enhancement of PVRL2 Fc binding in thepresence of CPA.7.002 and CPA.7.005 antibodies which we did not observewhen HEK cells were used as targets.

NK cell cytotoxicity assay with Reh cells: The first target weinvestigated in the NK cell cytotoxicity assay was the Reh line. Reh wasinitially selected as it showed robust levels of PVRL2 by flowcytometry, but a low frequency of other activating ligands such as NKG2Dligands, and low expression of PVR (FIGS. 94A-94H). Traditional NK celltargets were not used, such as K562, due to their expression of a highfrequency of NKG2D ligands, and high expression of PVR, which may mask afunctional effect of the PVRIG antibodies. Importantly, Reh cells didnot express any NK cell receptors known to interact with PVRL2 and PVRsuch as TIGIT, DNAM-1, and PVRIG.

Upon screening our panel of PVRIG antibodies in this assay, we foundfour antibodies that were able to modulate NK cell-mediated cytotoxicity(FIG. 99). These four antibodies were those that were discussed in thebiochemical assay results section-CPA.7.002, CPA.7.005, CPA.7.021, andCPA.7.050. In all cases, addition of these antibodies enhanced NKcell-mediated cytotoxicity against Reh cells (FIGS. 95a-95c ). Additionof CPA.7.002 and CPA.7.005 enhanced cytotoxicity most robustly (FIGS.95a-95b ), followed by CPA.7.021 and CPA.7.050 which showed similarlevels of enhancement (FIG. 95c ). FIG. 95d shows aconcentration-dependent analysis of enhancement of NK cell-mediatedcytotoxicity by CPA.7.002 and CPA.7.021. Blocking antibodies towardsreceptors that have been reported to also bind PVRL2 such as TIGIT andDNAM-1 were added to the assay with Reh cells as comparators. As shownin FIGS. 95a-95f , the addition of TIGIT and DNAM-1 antibodies did notshow functional effects in this assay.

NK cell assay with MOLM-13 cells: To assess whether PVRIG antibodieswere able to modulate NK cell-mediated cytotoxicity against a secondtarget, MOLM-13 cells were utilized. MOLM-13 also express PVRL2analogous to Reh cells, but also have robust expression of PVR (FIGS.94A-94H). Like the Reh cells, MOLM-13 did not express any NK cellreceptors. Utilization of this cell line, in addition to Reh cells,would indicate whether PVRIG antibodies can modulate NK cell-mediatedcytotoxicity in the context of different receptor-ligand interactions,particularly when PVR is expressed.

Upon screening our PVRIG antibodies in this assay, we found that thefunctional effect of CPA.7.021 was diminished and did not showsignificant enhancement of NK cell-mediated cytotoxicity above controllevels (FIG. 97a ). In contrast, CPA.7.002 and CPA.7.005 were able toenhance NK cell-mediated cytotoxicity in this assay (FIG. 97a ). Using acomparator antibody, blockade of TIGIT did not show functional effectsin this assay when compared to control (FIG. 97b ).

Summary and Conclusions

Using our antibody phage platform, we generated a panel of antibodiesagainst the human PVRIG antigen that showed an ability to block theinteraction of PVRIG with its ligand PVRL2, and enhance NK cell-mediatedcytotoxicity against two hematological cell lines. The ability of thePVRIG antibodies to inhibit PVRIG and PVRL2 interaction was influencedby the orientation of the assay as well as pre-incubation steps,representative of potential antibody dynamics with PVRIG inphysiological settings such as cancer. Four antibodies showed an abilityto enhance NK cell-mediated cytotoxicity against the Reh cell line, butonly two antibodies showed an ability to enhance cytotoxicity againstMOLM-13 cells. This difference may be attributed to the alternatereceptor-ligand interactions involved in NK cell-mediated recognition ofeach cell line, and/or differential properties of the antibodies andtheir potency in modulating the function of PVRIG.

Example 26. Effect of Anti-PVRIG Antibodies on GD T Cell ActivationUsing PVRL2 Ectopic or Naturally Expressing Cells

A cell based assay is used to test the effect of anti-PVRIG antibodieson gamma delta T cell activation, proliferation and cytokine secretion.Purified human gamma delta T cells are activated with HMBPP or IPP andco-cultured with target cells (e.g. REH, MOLM-13)_that naturally expressPVRL2 or with target cells ectopically expressing PVRL2 or empty vector(e.g. CHO, Raji, 721.221). Gamma delta T cell function is assessed byexamining cytokine production (e.g. IFN-γ, IL-17)_in culturedsupernatants or cytotoxic activity on the target cells. PVLR2 expressionis expected to have a basal stimulatory effect on gamma delta T cellactivation, mediated through endogenous DNAM1—a known costimulatorycounterpart receptor of PVRL2 on gamma delta T cells. In the presence ofantagonistic anti-PVRIG Abs, cytokine production or cytotoxic activityis expected to be further enhanced, due to their blocking of theinhibitory function of endogenous PVRIG on gamma delta T cellactivation. Accordingly, agonistic anti-PVRIG Abs are expected to showinhibition of gamma delta T cell activation.

Example 27: Effect of Proteins on Human T Cells Activated Using Anti-CD3and Anti-CD28 in the Presence of Autologous PBMCs

Materials and Methods

In these experiments the effects of PVRIG on human T cells which wereactivated using anti-CD3 and anti-CD28 in the presence of autologousPBMCS is evaluated. Conversely, this assay can also be used to assay theeffects of anti-PVRIG antibodies on T cell activation.

PVRIG hECD-hIg fusion protein (FIG. 92BA), composed of the ECD of humanPVRIG fused to the Fc of human IgG1 bearing C220, C226 and C229 to Smutations at the hinge, was produced at GenScript (China) by transienttransfection in CHO-3E7 cells which were cultured for 6 days, followedby protein A purification of cell harvest. The final product wasformulated in PBS pH 7.2. Expression vector used was MammalianExpression Vector pTT5, in which PVRIG gene is driven by CMV promoter.

CD4+ Human T cell Isolation Kit II is purchased from Miltenyi (Cat.#130-094-131). hIgG1 control (Synagis®) is obtained from Medimmune Inc.Anti-human CD3 Ab (OKT3, Cat #16-0037) and anti-human CD28 Ab (cloneCD28. 2; Cat #16-0289) are purchased from eBioscience. Dynabeads M-450Epoxy (Cat. #140. 11) are purchased from Invitrogen. Buffy coats ofhuman blood are obtained from LifeSource. Ficoll-Paque Plus (Cat.#17-1440-02), is purchased from GE HealthCare.

Isolation of PBMCs from buffy coats using Ficoll separation: Total PBMCsare suspended in Ex-Vivo 20 medium, and irradiated at 3000 rad. NaïveCD4+ T cells are isolated from buffy coats of three healthy humandonors' blood using CD4+ Human T cell Isolation Kit II (Miltenyi)according to manufacturer's instructions and co-cultured with irradiatedautologous PBMCs at a ratio of 1:1 (1. 5×10⁵ T cells with 1. 5×10⁵irradiated PBMCs per well). The cultures are activated with anti-CD3 (0.5 μg/ml) and anti-CD28 (0. 5 μg/ml) antibodies. Either an anti-PVRIGantibody or a PVRIG ECD protein are added to the culture at theindicated concentrations. After 24 hr in culture, cells are pulsed withH3-thymidine. Cells are harvested after 72 hours in culture.

For the ECD experiment, the results are expected to cause a dosedependent inhibition of T cell proliferation and/or activation,supporting the therapeutic potential of immunoinhibitory PVRIG basedtherapeutic agents (e.g. PVRIG polypeptides or PVRIG fusion proteinsaccording to at least some embodiments of the invention) for treating Tcell-driven autoimmune diseases, such as rheumatoid arthritis, multiplesclerosis, psoriasis and inflammatory bowel disease, as well as fortreating other immune related diseases and/or for reducing theundesirable immune activation that follows gene or cell therapy.Essentially, immunoinhibitory PVRIG proteins that agonize PVRIG shouldprevent or reduce the activation of T cells and the production ofproinflammatory cytokines involved in the disease pathology of suchconditions.

In addition, these results are also expected to support a therapeuticpotential of immunostimulatory anti-PVRIG antibodies that reduce theinhibitory activity of PVRIG for treating conditions which will benefitfrom enhanced immune responses such as immunotherapy of cancer,infectious diseases, particularly chronic infections and sepsis.Essentially, immunostimulatory anti=PVRIG antibodies will promote theactivation of T cells and elicit the production of proinflammatorycytokines thereby promoting the depletion of cancerous or infected cellsor infectious agents.

Example 28: Inhibition of T Cell Activation Assay

In these experiments the effects of PVRIG ECDs or anti-PVRIG antibodieson T cell activation in a bead assay.

Materials & Methods

Isolation of human T Cells: Buffy coats are obtained from Stanford BloodBank from healthy human donors. CD3+ T cells are isolated from buffycoats using RosetteSep kit (StemCell Technologies) followingmanufacturer's instructions. Cells are analyzed with anti-CD45 andanti-CD3 by flow cytometry to evaluate the % of CD3+ cells obtained.Viability is evaluated after thawing prior to the assay.

Bead Coating and QC: Tosyl activated beads (Invitrogen, Cat #14013) at500×10⁶/ml are coated with anti-CD3 mAb and either PVRIG ECD proteins oranti-PVRIG antibodies in a two-step protocol: with 50 μg/ml humananti-CD3 clone UTCH1 (R&D systems, Cat # mab 100) in sodium phosphatebuffer at 37° C. overnight, followed with 0-320 μg/ml of either PVRIGECD proteins or anti-PVRIG antibodies for another overnight incubationat 37° C.

The amount of PVRIG protein (either ECD or antibody) bound to the beadsis analyzed.

Bead assay setup: 100 k human CD3+ T cells are cultured with 100 k or200 k beads coated with various concentrations of the PVRIG protein for5 days in complete IMDM (Gibco, Cat #12440-053) supplemented with 2% ABhuman serum (Gibco, Cat #34005-100), Glutmax (Gibco, Cat #35050-061),sodium pyruvate (Gibco, Cat #11360-070), MEM Non-Essential Amino AcidsSolution (Gibco, Cat #11140-050), and 2-mercaptoethanol (Gibco, Cat#21985). At the end of 5 day culture, cells are stained with anti-CD25,anti-CD4, anti-CD8, and fixable live dead dye to determine CD25expression levels on each subset of cells. Supernatants are collectedand assayed for IFNγ secretion by ELISA (Human INFγ duoset, R&D systems,DY285).

In these experiments human CD3 T cells co-cultured with beads coatedwith various concentration of PVRIG-protein are analyzed for their levelof expression of CD25. Both CD4+ and CD8+ cells are anticipated to showdose dependent inhibition by the PVRIG-ECD-fusion protein, or,conversely, both CD4+ and CD8+ cells are anticipated to show dosedependent activation by the PVRIG-antibody.

Example 29: Epitope Mapping of Anti-Human PVRIG Antibodies Based onCynomolgus Cross-Reactivity Rationale and Objectives

The objective of this study is to identify the epitopes on the PVRIGprotein that determine cross-reactivity of anti-human PVRIG antibodiesagainst the cynomolgus monkey (cyno) orthologue. Many of the leadantibodies against human PVRIG target show varied degrees of cynocross-reactivity despite the fact that many of these antibodies belongto the same epitope bin. To shed light on the molecular basis ofhuman/cyno cross-reactivity (or lack thereof), several cyno-to-humanmutations of the PVRIG recombinant proteins were designed, expressed andpurified, and tested for binding to a panel of anti-human PVRIGantibodies in ELISA.

Methods

Design of cyno-to-human PVRIG variants: Sequence alignment of human andPVRIG extracellular domains (ECDs) shows 90% sequence identity and 93%sequence homology between human and cyno orthologs (FIG. 100). Based onthe nature of the mutations (conserved vs non-conserved) and thesecondary structure prediction (coil vs extended) of the mutationregion, three site-directed mutants of the cyno PVRIG were designed toprobe the cyno-cross reactivity focused epitope mapping. These mutantsinclude H61R, P67S, and L95R/T97I cyno PVRIG. Wild type cyno and humanPVRIG were also generated.

Expression and purification of cyno, human, and hybrid PVRIG variants:All the PVRIG variants were expressed as ECD fusions with a C-terminal6×His tag (SEQ ID NO: 7) in mammalian cells. The proteins were purifiedby affinity purification, ion-exchange chromatography, andsize-exclusion chromatography. The purified proteins werebuffer-exchanged into PBS buffer (pH 7.4) and stored at 4° C.

ELISA to determine PVRIG-antibody interaction: The functional ELISA wasperformed as follows: cyno, human, and cyno/human hybrid PVRIG(His-tagged) recombinant proteins were adsorbed on an IA plate overnightat 4° C. Coated plate wells were rinsed twice with PBS and incubatedwith 300 μL blocking buffer (5% skim milk powder in PBS pH 7.4) at roomtemperature (RT) for 1 hr. Blocking buffer was removed and plates wererinsed twice more with PBS. Plate-bound PVRIG variants were incubatedwith anti-human PVRIG mAbs (human IgG1 isotype) in solution (linearrange of 0.1 μg/mL to 8 μg/mL in a 50 μL/well volume) at RT for 1 hr.Plates were washed three times with PBS-T (PBS 7.4, 0.05% Tween20), thenthree times with PBS and 50 μL/well of a HRP-conjugated secondaryantibody was added (Human IgG Fe domain specific, JacksonImmunoResearch). This was incubated at RT for 1 hr and plates werewashed again. ELISA signals were developed in all wells by adding 50 μLof Sureblue TMB substrate (KPL Inc) and incubating for 5-20 mins. TheHRP reaction was stopped by adding 50 μL 2N H2SO4 (VWR) and absorbancesignals at 450 nm were read on a SpectraMax (Molecular Devices) orEnVision (PerkinElmer) spectrophotometer. The data were exported toExcel (Microsoft) and plotted in GraphPad Prism (GraphPad Software,Inc.).

Results

S67, R95, and 197 residues as determinants of cyno cross-reactivity: Thebinding data shown in FIG. 101 clearly shows that the S67, R95, and 197residues affect the cyno cross-reactivity of various antibodies. Whilethe P67S cyno-to-human mutation negatively impacts the binding ofCPA.7.002 and CPA.7.041, the L95R/T971 cyno-to-human mutationsignificantly improves the binding of CPA.7.002, CPA.7.021, CPA.7.028,and CPA.7.041. On the other hand, H61R cyno-to-human mutation does notaffect the binding of any of the antibodies tested.

Relative binding to cyno-to-human variants suggests three epitopegroups: The relative binding of the antibodies to cyno, human and hybridPVRIG variants suggests 3 distinct epitope groups: Group 1 binds toR95/I97 residues (CPA.7.021 and CPA.7.028). Group 2 binds to S67 andR95/I97 residues (CPA.7.002 and CPA.7.041). Group 3 does not bind to S67or R95/I97 residues (CPA.7.024 and CPA.7.050). The epitope groups showstrong correlation to the degree of cyno cross-reactivity of theseantibodies (FIG. 102).

Summary and Conclusions

The restricted epitope mapping based on cyno-to-human variations in thePVRIG ECD identified S67, R95, and 197 residues as determinants of cynocross-reactivity of anti-human PVRIG antibodies. The completerestoration of binding to L95R/T971 cyno PVRIG for CPA.7.021 andCPA.7.028 antibodies and improved binding of CPA.7.002 to this mutantstrongly suggests that R95 and 197 residues are critical human PVRIGepitopes for these antibodies. These findings also suggest a possibleway to predict cross-reactivity to non-human primate PVRIG orthologsbased on their primary amino acid sequence.

1. A method of activating T-cells of a patient with cancer comprisingadministering an anti-PD-L1 antibody and an anti-PVRIG antibody to saidpatient, wherein said anti-PVRIG antibody comprises: i) a heavy chainvariable domain comprising the vhCDR1, vhCDR2, and vhCDR3 from SEQ IDNO:1434 and ii) a light chain variable domain comprising the vlCDR1,vlCDR2, and vlCDR3 from SEQ ID NO:1453, wherein a subset of said T-cellsof said patient are activated.
 2. The method according to claim 1wherein said anti-PVRIG antibody comprises the heavy chain variabledomain of SEQ ID NO:1434 and the light chain variable domain of SEQ IDNO:1453.
 3. The method according to claim 2 wherein said anti-PVRIGantibody comprises the CH1-hinge-CH2-CH3 region from IgG1, IgG2, IgG3,or IgG4, wherein said hinge region optionally comprises mutations. 4.The method according to claim 3 wherein said anti-PVRIG antibodycomprises the CL region of human kappa 2 light chain.
 5. The methodaccording to claim 1 wherein said T-cells are cytotoxic T-cells (CTLs).6. The method according to claim 1 wherein said T-cells are selectedfrom the group consisting of CD4⁺ T-cells and CD8⁺ T-cells.
 7. Themethod according to claim 1 wherein said activation is measured as anincrease in interferon-γ production and/or an increase in cytokinesecretion.
 8. A method of activating T-cells of a patient with cancercomprising administering an anti-PD-L1 antibody and an anti-PVRIGantibody to said patient, wherein said anti-PVRIG antibody comprises: i)a heavy chain variable domain comprising the vhCDR1, vhCDR2, and vhCDR3from SEQ ID NO:1447 and ii) a light chain variable domain comprising thevlCDR1, vlCDR2, and vlCDR3 from SEQ ID NO:1462, wherein a subset of saidT-cells of said patient are activated.
 9. The method according to claim8 wherein said anti-PVRIG antibody comprises the heavy chain variabledomain of SEQ ID NO:1447 and the light chain variable domain of SEQ IDNO:1462.
 10. The method according to claim 9 wherein said anti-PVRIGantibody comprises the CH1-hinge-CH2-CH3 region from IgG1, IgG2, IgG3,or IgG4, wherein said hinge region optionally comprises mutations. 11.The method according to claim 10 wherein said anti-PVRIG antibodycomprises the CL region of human kappa 2 light chain.
 12. The methodaccording to claim 9 wherein said T-cells are cytotoxic T-cells (CTLs).13. The method according to claim 9 wherein said T-cells are selectedfrom the group consisting of CD4+ T-cells and CD8+ T-cells.
 14. Themethod according to claim 9 wherein said activation is measured as anincrease in interferon-γ production and/or an increase in cytokinesecretion.
 15. A method of activating T-cells of a patient with cancercomprising administering an anti-PD-L1 antibody and an anti-PVRIGantibody to said patient, wherein said anti-PVRIG antibody comprises: a)a heavy chain variable domain comprising: i) a vhCDR1 comprising SEQ IDNO:885; ii) a vhCDR2 comprising SEQ ID NO:886; iii) a vhCDR3 comprisingSEQ ID NO:887; and b) a light chain variable domain comprising: i) avlCDR1 comprising SEQ ID NO:889; ii) a vlCDR2 comprising SEQ ID NO:890;iii) a vlCDR3 comprising SEQ ID NO:891, wherein a subset of said T-cellsof said patient are activated.
 16. The method according to claim 15wherein said anti-PVRIG antibody comprises the CH1-hinge-CH2-CH3 regionfrom IgG1, IgG2, IgG3, or IgG4, wherein said hinge region optionallycomprises mutations.
 17. The method according to claim 16 wherein saidanti-PVRIG antibody comprises the CL region of human kappa 2 lightchain.
 18. The method according to claim 15 wherein said T-cells arecytotoxic T-cells (CTLs).
 19. The method according to claim 15 whereinsaid T-cells are selected from the group consisting of CD4⁺ T-cells andCD8⁺ T-cells.
 20. The method according to claim 15 wherein saidactivation is measured as an increase in interferon-γ production and/oran increase in cytokine secretion.
 21. A method of activating T-cells ofa patient with cancer comprising administering an anti-PD-L1 antibodyand an anti-PVRIG antibody to said patient, wherein said anti-PVRIGantibody comprises: a) a heavy chain comprising: i) aVH-CH-hinge-CH2-CH3, wherein the VH is SEQ ID NO:1434 and wherein theCH1-hinge-CH2-CH3 region is from IgG4; and b) a light chain comprising:i) a VL-CL, wherein the VL is SEQ ID NO:1453 and wherein the CL regionis from human kappa 2 light chain.
 22. The method according to claim 21wherein said hinge region optionally comprises mutations.
 23. A methodof activating T-cells of a patient with cancer comprising administeringan anti-PD-L1 antibody and an anti-PVRIG antibody to said patient,wherein said anti-PVRIG antibody comprises: a) a heavy chain comprising:i) a VH-CH1-hinge-CH2-CH3, wherein the VH is SEQ ID NO:1447 and whereinthe CH1-hinge-CH2-CH3 region is from IgG4; and b) a light chaincomprising: i) a VL-CL, wherein the VL is SEQ ID NO:1462 and wherein theCL region is from human kappa 2 light chain.
 24. The method according toclaim 23 wherein said hinge region optionally comprises mutations.